Search

Wetlands are highly sensitive to pollution effects as they are usually low lands in comparison to the surroundings. Consequently, pollution with heavy metals in the wetlands ecosystems may have changed the ecosystem's biogeochemistry. Gomishan and Zarivar wetlands are regarded as two of the most important wetland regions in south eastern corner of Caspian Sea and in Western Iran respectively. We report the results from a detailed research based on two metals (Pb and Cd) measured in muscle of two fish species (Cyprinus carpio and Liza saliens) from Gomishan and Zarivar wetlands. Twenty three leaping mullet (Liza saliens) fish samples (from the 9 stations) and twenty common carp (Cyprinus carpio) were collected in year 2013 from Gomishan international wetland and Zarivar wetland respectively. Using a hot-block digester, the muscle samples were digested with a combination of perchloric acid (HCLO4) and nitric acid (HNO3) and then examined by atomic absorption spectrophotometry. The ranges of the concentrations of metals in the edible muscle tissues were: Pb 0.14–0.57 μg/g; Cd 0.34–0.49 μg/g; in common carp and Pb 0.00075–0.03 μg/g; Cd; 0.08–0.025 μg/g in leaping mullet. Pb and Cd concentrations between both fish species were statistically significant (insert the p value). There was no statistically significant correlation between the metal concentration of the muscles and the fish weight for both species. The concentrations of heavy metals in muscle of examined both fish species were below the permissible levels and should not pose health problems for human consumption. Periodic monitoring of these metals in both fishes and water to ensure safety is recommended.

This study assesses the frequency, distribution, characteristics, and chemical composition of microplastics (MPs) in the gut of highly consumed fish species, namely leaping mullet (Chelon saliens), common carp (Cyprinus carpioi), and Caspian kutum (Rutilus caspicus), in the southern Caspian Sea biome. Fibers are found to be the only shape of MPs. Black MPs and polystyrene, polypropylene, and polyethylene terephthalate polymers are dominant. MP frequency is highest in leaping mullet's gut, while kutum specimens exhibited the lowest MP frequency, reflecting that leaping mullet is a neritic species and thus highly exposed to MP influx in shallow coastal water, while the other species are benthopelagic. The estimated condition index reflected a significant difference between the species, implying that MPs may pose adverse health impacts on leaping mullet and common carp, with no undesirable effect on Caspian kutum. No significant relationship exists between biological parameters and the MP frequency in the fish gut.

Members of the phylum Myxozoa are among the most cosmopolitan parasites of fish, and the genus Myxobolus is the largest within the class Myxosporea in freshwater and marine environment from all over the world. Myxobolus parvus has been reported from mainly mugilid fishes from different localities including the Black Sea. Leaping mullet Liza saliens collected by fishermen off Sinop coasts of the Black Sea in the period between September 2014 and December 2015 was investigated for myxosporean parasites using conventional methods. M. parvus Schulman, 1962 was the only Myxobolus species identified and it was found in the gills, gall bladder, kidney tubules, and inside several cysts located on the lower jaw of examined fish in the present study. Morphometric data and all details of parasitic infection were determined and presented in tables and illustrated in figures. Molecular characterization was also conducted to reveal its position within Myxosporea. Two M. parvus samples from gills and kidney showed the same haplotype and appeared in the same sub-lineage with marine Myxobolus species. The closest haplotypes to M. parvus were Triactinomyxon sp. and Endocapsa sp. which are the actinosporeans forms of unknown myxozoan species. These data are the first in molecular characterization of M. parvus, its occurrence in lower jaw of leaping mullet along with its infection prevalence values off the Turkish Black Sea coasts.

Betanoda virus is an emerging problem in several marine fish species in various geographic areas all over the world. In recent years, mullets stock of the Caspian Sea decreased dramatically and a betanoda virus like-agent was introduced as the cause of mullet’s mortality. The main objective of the present study is to compare hemato-biochemical parameters in healthy and infected sharpnose mullets (Liza saliens) to betanoda virus like-agent. The adult sharpnose mullets (34 clinically affected fish + 34 apparently healthy fish) were captured from the southeast the Caspian Sea from 2012 to 2013. All of the captured fish were 4 or 5 years old. The main clinical sings of infected fish were lethargy, severe abdominal distention, abnormal swimming and hyperinflation of swim bladder. The results showed that the weight and total length of infected fish were significantly lower than the healthy fish. The results of histopatological evaluation and indirect florescent antibody test were confirmed the presence of a betanoda virus-like agent in infected fish. The diseased fish were severely anemic (hypochromic macrocytic anemia) and had a serious haemopoietic disorders. The anemia associated with a sever leukopenia, a significant rise of neutrophils and immature neutrophils and a significant decries of lymphocytes percentage. Total protein, albumin and total immunoglobulin levels were significantly reduced in the serum of infected fish, while the activities of aspartate aminotransferase and alanine aminotransferase significantly increased when compared to the healthy fish. These results suggested that the feeding of the infected fish to betanoda virus like-agent were disturbed and it could be cause haemato-biochemical disorder and mortality of the fish in Iranian water of the Caspian Sea.

Quercetin, resveratrol, naringenin, hesperidin, and rutin are phenolic compounds/flavonoids that may have roles in the reduction of cancer susceptibility. In this study, in vitro modulatory effects of them were studied on liver CYP1A1 associated 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione S-transferase (GST) activity of leaping mullet (Liza saliens). All of the phenolic compounds/flavonoids used exerted an inhibitory effect on both EROD and GST activities of fish. Quercetin, resveratrol, hesperidin, and rutin were found to inhibit EROD activity in a competitive manner; on the other hand, naringenin was found to inhibit EROD activity in a noncompetitive manner. Ki values of quercetin, resveratrol, naringenin, hesperidin, and rutin were calculated from Dixon plots as 0.12 μM, 0.67 μM, 2.63 μM, 18 μM and 0.1 mM, respectively. Resveratrol, quercetin, and hesperidin were found to inhibit GST activity in a competitive manner; on the other hand, rutin and naringenin were found to inhibit GST activity in a mixed-type manner. Ki values of resveratrol, quercetin, hesperidin, naringenin, and rutin were calculated from Dixon plots as 3.2 μM, 12.5 μM, 45 μM, 128 μM, and 150 μM, respectively. The results suggest that quercetin and resveratrol containing foods are effective in the prevention and treatment of cancer.

During examination of some species of the family Mugilidae, two coelozoic myxozoans were observed in the gall-bladder of the leaping mullet, Liza saliens (Risso). Spore morphology allowed us to allocate them to the genus Ellipsomyxa Koie, 2003, one of which is described here as new. Ellipsomyxa kalthoumi n. sp. was observed forming globular pseudoplasmodia and free spores floating in the bile. Mature spores are ellipsoidal, measuring 13–21 × 10–15 (17.2 × 13.2) µm and possessing two equal spherical polar capsules, 5–6 (5.5) µm in diameter, opening subterminally in opposite directions, with nine polar filament coils. Morphological data and molecular analysis of the small subunit rDNA sequences helped identify this parasite as a new species of Elliposmyxa. The second species identified as E. mugilis (Sitja-Bobadilla & Alvarez-Pellitero, 1993) has oval spores with rounded ends, measuring 10–11 × 7–9 (10.5 × 8.0) µm and possessing two polar subspherical capsules, 2.7–3.0 (2.8) µm in diameter, opening subterminally in opposite directions. Spore morphometry and molecular study of the small subunit (SSU) of the rRNA gene identified this species as E. mugilis described from the same host in the Western Mediterranean off Spain. Phylogenetic analysis revealed Elliposmyxa as a monophyletic clade and showed that E. mugilis, E. syngnathi Koie & Karlsbakk, 2009, E. adlardi Whipps & Font, 2012 and E. gobii Koie, 2003 group in a subclade containing the Tunisian isolate of E. mugilis whereas E. kalthoumi n. sp. appears in a second subclade together with four Australian species, E. maniliensis Heiniger & Adlard, 2014, E. apogoni Heiniger & Adlard, 2014, E. nigropunctatis Heiniger & Adlard, 2014 and E. arothroni Heiniger & Adlard, 2014.

The concentrations of Cr, Cu, Fe, Mn, Ni, Pb, Cd, and Zn were determined in the brain, heart, liver, gill, gonad, spleen, kidney, and red and white muscles of Liza saliens (leaping mullet). Trace element levels in fish samples were analyzed by flame atomic absorption spectrometry. Among the non-essential metals, the levels of Ni and Pb in the tissues were higher than limits for fish proposed by FAO/WHO, EU, and TFC. Generally, the levels of the non-essential metals were much higher than those of manganese in the red and white muscles. Fe distribution pattern in tissues was in order of spleen > liver > heart > gill > brain > kidney > gonad > red muscle > white muscle. Red muscle was not within the safe limits for human consumption because non-essential metal (Ni, Pb) contents were higher than standard limits.

Pollution of the aquatic environment is a global concern owing to the devastating effects of contaminants whose levels are growing at an alarming rate, and it has become a major threat for marine organisms, as well as to humans as consumers. This study has been carried out on leaping mullet obtained from Aliaga Bay, which is located on the west coast of the Aegean Sea near Izmir and hosts the world's fifth largest shipyard, plus a broad range of industrial activities, including an oil refinery and a paper factory. The waste from these industries, combined with municipal sewer discharges, is the main cause of pollution in this region. There is no national documentation or research on the determination of pollution resulting from the industrial activities in this area. In the present study, the degree of induction of CYP4501A-associated 7-ethoxyresorufin O-deethylase (EROD) activity and immunochemical detection of CYP1A1 in the liver of leaping mullet (Lisa saliens) were used as biomarkers for the assessment of polycyclic aromatic hydrocarbon (PAH)-type organic pollutants in Aliaga Bay. Mullet caught from different locations of the bay had approximately 52 times more EROD activity than the feral fish sampled from a clean reference site near Foca, Izmir. The results of this study indicate that Aliaga Bay is highly contaminated with PAH-type organic pollutants. © 2009 Springer Science+Business Media B.V.

Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 μM and 3 μM, respectively. The Lineweaver–Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The Ki values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 μM, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:340–3347, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20200

Bu çalışmada Kahramanmaraş Sır Baraj Gölü'nde yaşayan kefal (Liza abu) bazı biyolojik özellikleri araştırılmıştır. Kasım 2014 – Eylül 2015 tarihleri arasında gerçekleştirilen araştırmada, örneklenen toplam 301 birey incelenmiştir. Örnekler 44, 52, ve 56 mm göz açılarak daki ağırlık yakalanmiştir. Örnekler barajın 4 ayrı noktasından avlanmıştır. Avcılık, fanyalı ağlar ile yapılmıştır.Örnekleren bireylerin boy ve ağırlıkları belirlendikten sonar karın kısımları açılarak cinsiyetleri belirlenmiş ve gonad ağırlıkları tartılmıştır. İncelen bireylerin yaşları tayinleri yapıldıktan sonra boy ve ağırlık çatal büyümeleri, yaş-boy ilişkisi, yaş-ağırlı kilişkisi, kondisyon faktörü, üreme dönemi, cinsiyet oranı ve morfolojisi incelenmiştir.Maksimum beş yaşın akadar birey saptanmış ve bire ylerin çatal boy dağılımı 12.00-24.00 cm arasında, ağırlığı ise 25.46-186.83 g arasında değişim göstermiştir. Erkek, dişi ve tüm bireyler için Sırasıyla boy-ağırlık ilişkisi W=0.009*L3.144, W=0.014*L3.018 ve W=0.010*L3.095 bulunmuştur. Von Bertalanffy büyüme parametreleri erkekler için L∞= 18, 75 cm, k= 0.32 yıl-1, t0= -0.88 yıl olarak, dişiler için L∞= 26.90 cm, k= 0.21 yıl-1, t0= -1.61 yıl olarak ve tüm bireyler için L∞= 23.65 cm, k= 0.21 yıl-1, t0= -1.54 yıl hesaplanmıştır.Kondisyon faktörleri Mart'ta maksimum (K=2.41) iken Şubat'ta minimum (K=1.21) bulunmuştur. Gonadosomatik indeks değerlerine gore üreme sezonunun Mart ayında başladığı ve Ağustos ayınakadar devam ettiği belirlenmiştir.Anahtar Kelimeler: Sır Baraj Gölü, Ceyhan nehrin, Liza abu, yaş, büyüme, üreme. In this study, some biological characteristics of Liza abu which lives in Sır Reservoir of Kahramanmaraş (Turkey) were examined. The studies were conducted between Desember 2014 and September 2015, and totally 301 fish samples were collected by gillnets with mesh sizes 44, 52 and 56 mm. The samples were taken from four different points of the Sır Reservoir with the aid of gill nets. Fork lengths, body and gonad weights of samples were measured, sexes determined, and age determined by using scales. Length and weight growth depend on age, age-length and age-weight relations, condition factor, gonadosomatic index, sex ratio, and morphology of the collected samples were studied.The maximum age group was determined as 1 and 5, and the specimen's fork length between 12.00 and 24.00 cm, and weight between 25.46 and 186.83 g. The length-weight relationships were found asW = 0.009*L3.144 for males, W= 0.014*L3.018 for females and W = 0.010*L 3.095for all individuals. Von Bertalanffy growth parameters were estimated as L∞= 22.66 cm, k= 0.32 year-1, t0= -0.88 year for males, L∞= 26.90 cm, k= 0.21 year-1, t0= -1.61 year for females and L∞= 23.65 cm, k= 0.21 year-1, t0= -1.54 year for all population.The average condition factors were found as maximum in March (K=2.41) and as minimum in February (K=1.21). According to the gonadosomatic index values spawning season take place from the beginning of March until August 2015.Key words: Sır Reservoir, Ceyhan River, Liza abu, age, growth, reproduction. 69

The purpose of the present study was to elucidate in vitro effects of Hg(2+), Zn(2+), Ni(2+) and Cd(2+) on cytochrome P4501A1 (CYP1A1) dependent EROD activities in leaping mullet liver microsomes. Fish captured from the most polluted part of Izmir Bay, had highly elevated EROD activities, and induced CYP1A1 protein levels as determined by Western blotting. Although all of the metal ions caused inhibition of the initial velocity of the reaction, Hg(2+) and Cd(2+) exhibited much higher inhibitory effect at lower concentrations and they were evidently more potent inhibitors than others. The inhibitor concentration giving 50% inhibition (IC(50) values) of Zn(2+), Ni(2+), Cd(2+) and Hg(2+) of initial EROD activity were 107, 16, 1.3 and 0.15 micromolar, respectively. Glutathione (GSH) at 0.5 mM final concentration, completely reversed Ni(2+) and Cd(2+) inhibition of EROD activity indicating the protective action of GSH.

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 ± 0.22 nmol formaldehyde formed/min/mg protein (mean ± SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver–Burk plot, two Km values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 μM concentrations of these inhibitors. At 100 μM final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 μM final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 μM. IC50 values were calculated to be 20 μM for ketoconazole, 48 μM for cimetidine (both specific P4503A inhibitors), 164 μM for quinidine (P4502D inhibitor), and 59 μM for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:169–176, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10075

The present study was conducted on 428 moribund mullet fish samples to isolate and identify the causative agent of a mysterious acute mortality which recently occurred in wild mullets in Iranian waters of Caspian Sea, suspected to be due to viral nervous necrosis (VNN) disease. Disease investigation was carried out employing various diagnostic procedures such as virology, bacteriology, parasitology, haematology, histopathology, IFAT, IHC and nested RT-PCR. Brain and eye samples of affected fishes were collected in sterile conditions and then kept at -80 °C for cell culture isolation and nested RT-PCR detection of the causative agent. Other tissue samples were also collected and fixed for histopathology, IHC and EM examinations. CPE was observed in cell cultures at 6 days after inoculation. Nine samples were found positive with virological assay. Nested RT-PCR, performed on suspected tissues and CPE positive samples, showed that about 21 tissue samples and all the CPE positive samples were positive for VNN virus (VNNV). IFAT was selected as a confirmatory method for detecting the presence of Betanodavirus antigen, cell culture isolation results and nested RT-PCR findings. Moreover, VNNV particles with 25-30 nm in diameter were also visualized in the infected brain and retina. In pathogenicity studies, guppy fishes bathed in VNNV-infected tissue culture (10-4 TCID50) showed clinical signs similar to naturally infected mullet after 15 days post infection (dpi), with mortality rates reaching up to 100% at 30 dpi. Affected organ samples as examined by cell culture isolation, IFAT, IHC and histopathology, revealed the presence of VNNV in the guppy fishes. In conclusion, it was confirmed that VNNV was the main causative agent for the disease outbreak in mullet fish in the Caspian Sea, and this is such first official report of VNN disease from Iran.

Bu çalışmada Çıldır gölündeki Cyprinus carpio (Linnaeus, 1758), Capoeta capoeta (Güldenstadt, 1773) ve Squalius turcicus (De Filippi, 1865) türlü balıkların bazı fenotopik özellikleri belirlenmiştir.Her bir balıkta standart boy (SL), sırt yüksekliği (SY), baş uzunluğu (BU), toplam vücut ağırlığı (W), pul ağırlığı (PA), yüzgeç ağırlığı (YA), iç organ ağırlığı (İOA), kılçıklı karkas ağırlığı (KA), ovaryum ağırlıkları (OA) ve pul yaşı belirlendi. Ayrıca kondüsyon faktörü (K), SL/SY, SY/SL, BU/SL, BA/W, YA/W, PA/W, karkas verimi, visserosomatik indeks (VSİ), gonadosomatik indeks (GSİ) oranları da hesaplanmıştır.Sazan ve kefal grubunda grup içi ve cinsiyete göre değerlendirildiğinde hem SL, BU, W, BA, KA, PA hem de SL/SY, SY/SL, BU/SL, BA/W, PA/W; YA/W, VSİ, KV, GSİ ve K faktörü bakımından istatistiki olarak önemli bir fark bulunmamıştır (P> 0,05). Karabalık grubunda ise SL (P0,05).Sonuç olarak, K faktörü düşükten büyüğe doğru karabalık, sazan ve kefal şeklinde iken KV açısından karabalık, kefal ve sazan sıralaması tespit edilmiştir. In this study, some phenotypic characteristics of the Cyprinus carpio (Linnaeus, 1758), Capoeta capoeta (Güldenstadt, 1773) and Squalius turcicus (De Filippi, 1865) species belongs to Çıldır Lake- Turkey were investigated.Each fish was determined for standard length (SL), back height (SY), head length (BU), total body weight (W), flake weight (PA), fins weight (YA), internal organ weight (İOA), carcass weight (KA), ovarian weights (OA) and the age flakes. In addition, condition factor (K), SL/SY, SY/SL, BU/SL, BA/W, YA/W, PA/W, carcass yield (KV), visserosomatic index (VSİ), gonadosomatic index (GSİ) rates were calculated.Compared to within the fish groups by gender, both the Cyprinus carpio group and the Squalius turcicus group, no statistically significant differences were obtained in terms of SL, BU, W, BA, KA, PA, SL/SY, SY/SL, BU/SL, BA/W, PA/W, YA/W, VSİ, KV, GSI and K factor (P>0,05). In the group Capoeta capoeta, there was a statistically significant difference, respectively; SL (P0,05).In conclusion, from small to large for K factor was found Capoeta capoeta, Cyprinus carpio and Squalius turcicus rankings, while Capoeta capoeta, Squalius turcicus and Cyprinus carpio rankings were determined in terms of KV. 65

This study continues a comparative cytogenetic analysis of the fish family Mugilidae, reporting the karyotype characterization of the leaping mullet, Liza saliens, by C-banding, Ag- and fluorochrome-staining, and completing the fluorescence in situ hybridization (FISH)-mapping of the 18S and 5S rRNA genes (rDNA) to the chromosomes of the six Mediterranean mullets, namely L. saliens, L. ramada, L. aurata, Mugil cephalus, Chelon labrosus and Oedalechilus labeo. In all species, except M. cephalus, the 5S rDNA sites were localized on a medium-sized acrocentric chromosome pair, which was considered homeologous in all of them. In L. saliens, an additional 5S rDNA site was detected in a location close to the one shown by major ribosomal genes in M. cephalus, i.e. the subtelomeric region of chromosome pair 1. The 5S rDNA site in M. cephalus is located on the smallest chromosome pair of the complement, which, on the other hand, though on a different position, bears 18S rDNA in all the species of Liza and Chelon examined. The heterochromatin composition and the major and minor ribosomal gene locations suggest that the karyotype of L. saliens (subgenus Protomugil) can be considered intermediate between the karyotype of the more primitive M. cephalus and those of the other Liza (subgenus Liza) species and of the representatives of the more derived genera Chelon and Oedalechilus.

A 2,037 bp CYP1A1 cDNA (GenBank AF072899) was cloned through screening of a λZipLox cDNA library constructed from the liver of a leaping mullet (Liza saliens) fish captured from Izmir Bay on the Aegean coast of Turkey using rainbow trout CYP1A1 cDNA as a probe. This clone has a 130 bp 5'-flanking region, a 1,563 bp open reading frame (ORF) encoding a 521-amino acid protein (58,972 Da), and a 344 bp 3'-untranslated region without a poly (A) tail. Alignment of the deduced amino acids of CYP1A1 cDNAs showed 58% and 69–96% identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1 cDNA was from a single-copy gene. Based on the comparison with CYP1A1 genes reported for fish and mammals, the leaping mullet CYP1A1 gene is probably split into 7 exons. The intron insertion sites were predicted. Alignment of the CYP1A1 cDNA encoded amino acids from 13 fish and 7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates. The possible associated secondary structure; conserved motifs and substrate-binding sites were discussed. The phylogenetic relationships of CYP1A1s among 13 fish species were analyzed by a distance method. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:243–255, 2001

In this study, the degree of induction of cytochrome P4501A-associated 7-ethoxyresorufin O-deethylase (EROD) activity and immunochemical detection of cytochrome P4501A in leaping mullet (Liza saliens) and common sole (Solea vulgaris) were used as biomarker for assessment of polycyclic aromatic hydrocarbon (PAH) or/and polychlorinated biphenyl (PCB) type organic pollutants along the Izmir Bay on the Aegean Sea coast, Turkey. Fish were captured in May 1995 and February and June 1996. Mullet caught from Pasaport, a highly urbanized and industrial section of the Bay, had approximately 62 times more EROD activity than the feral fish sampled from an uncontaminated site in the Outer Bay. Mullet caught along the pollutant gradient at the three other sites in the Bay exhibited less but highly significant induced EROD activity. An inverse relationship was found between the EROD activity in the fish and the distance between the catch point and the discharge region of polluted rivers and of industrial and domestic wastes into the Harbour. Studies using the polyclonal antibodies produced against mullet cytochrome P4501A showed a similar trend. In addition, EROD activities of benthic fish, common sole, captured from three different sites of the Bay also confirmed and extended the results obtained with those of mullet. Except that, common sole caught from site 5, the edge of Gediz river, exhibited higher EROD activity than that of fish caught from site 4, Tuzla. Although, detailed qualitative and quantitative analyses of organic chemicals in the waters and sediments of Izmir Bay are not available, the results of this study indicate that Inner and Middle Bays of Izmir Bay are highly contaminated with PAH and/or PCB type organic pollutants.

NADPH-cytochrome P450 reductase was purified to electrophoretic homogeneity from detergent-solubilized liver microsomes from the leaping mullet (Liza saliens). The purified reductase was characterized with respect to spectral, electrophoretic, and biocatalytic properties. In addition, effects of pH, ionic strength, and the substrate concentration on the NADPH-dependent cytochrome c reductase activity of the purified fish liver cytochrome P450 reductase were studied. Cytochrome P450 reductase was purified 438-fold with a yield of 17.5% with respect to the initial amount present in the fish liver microsomes. The specific activity of the enzyme was found to be 52.6 μmol cytochrome c reduced per minute per mg protein. The monomer molecular weight of the purified enzyme was calculated to be 77,000 ± 1000 when electrophoresed on polyacrylamide gels under the denaturing conditions in the presence of SDS. The absorption spectrum of fish reductase showed two peaks at 378 and 455 nm. NADPH-dependent cytochrome c reductase activity of the purified Liza saliens liver cytochrome P450 reductase was found to be maximal when pH was between 7.4 and 7.8. The apparent Km of the purified enzyme was found to be 7.69 μM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer, pH 7.7, at room temperature, and the enzyme was fully saturated by its substrate, cytochrome c, when the substrate concentration was at or above the 70 μM. Furthermore, the purified enzyme was biocatalytically active in reconstituting the 7-ethoxyresorufin O-deethylase activity in the reconstituted system containing purified mullet liver cytochrome P4501A1 and lipid. These results suggested that the purified fish liver cytochrome P450 reductase is similar to its mammalian counterparts with respect to spectral, electrophoretic, and biocatalytic properties. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 103–113, 1998

This paper reports in vitro effects of individual heavy metals (Cd(2+), Cu(2+) and Hg(2+)), and PAHs, including benzo[a]pyrene(BaP), indeno[1,2,3-cd]pyrene (IP) and fluoranthene (FL), and their mixtures on ethoxyresorufin-O-deethylase (EROD) activities using a plate-reader method. The results showed that all three metals inhibited EROD activity, while BaP/IP significantly induced the enzyme. However, FL alone decreased EROD activity. Moreover, co-treatment with BaP/IP and heavy metals inhibited PAH-induced EROD activities, while combined exposure to FL and heavy metals induced FL-inhibited EROD activities.

TEPE, Yalcin/0000-0002-8415-3754; Turkmen, Aysun/0000-0001-7461-4038; Turkmen, Mustafa/0000-0001-6700-5947; TEPE, Banu/0000-0002-3428-8167 WOS: 000297361100006 PubMed: 21822962 Present study was performed to examined the metal concentrations in muscle, liver, gonad and gill of gilthead seabream, European seabass, Leaping mullet and Flathead grey mullet from Paradeniz Lagoon, Mediterranean coastal area. The metal concentrations found in muscle tissue varied for Fe: 2.3-51.4, Zn: 6.63-14.8, Cu: 0.38-1.10, Mn: 0.38-1.06, Cr: 0.34-1.13, Ni: 0.58-.074, Pb: 0.32-1.02, Cd: 0.20-0.67 and Co: 0.30-0.61 mg/kg wet weight. Iron showed the highest levels in all tissues, and generally followed by zinc. On the other hand, cadmium, cobalt and lead showed lower levels than other metals. Statistically significant differences were observed in the mean metal values obtained from investigated fish species and their tissues. The levels of metals in the examined fish were well below the regulatory values of various govermental agencies. Scientific & Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [106Y024] Thanks to The Scientific & Technological Research Council of Turkey (TUBITAK) for its financial support (Project No: 106Y024) and also Kamil Yerdogan and Esra Aslan for their kind assistance in analysis.

WOS: 000299494300017 DANABAS, D., T. ALTUN and F. CELIK, 2011. The overwintering of leaping mullet (Liza saliens Risso, 1810) in fresh water. Bulg. J. Agric. Sci., 17: 846-850 The overwintering of leaping mullet (Liza saliens Risso, 1810) juveniles (average live weight (W) 5.20 +/- 0.02 g and average total length (L) 8.11 +/- 0.08 cm) was investigated, in this study. The trial was planned in 2 groups and triplicate. One of the trial groups (the Group 1) comprised of the fish stocked in ponds in the open area and the other group (the Group 2) of the fish stocked in the ponds covered with greenhouses. The average W and L values of the Group 2 after a period of 3 months were 8.70 +/- 0.04 g and 9.62 +/- 0.05 cm, respectively. The mortality rate (M) in this group was 0.95 +/- 0.1 % and 12.86 +/- 0.1 % in the Group 1. The difference among the W and L values, specific growth rates (SGR) and M averages obtained from the groups was found to be statistically significant (P

The overwintering of leaping mullet (Liza saliens Risso, 1810) juveniles (average live weight (W) 5.20±0.02 g and average total length (L) 8.11±0.08 cm) was investigated, in this study. The trial was planned in 2 groups and triplicate. One of the trial groups (the Group 1) comprised of the fish stocked in ponds in the open area and the other group (the Group 2) of the fish stocked in the ponds covered with greenhouses. The average W and L values of the Group 2 after a period of 3 months were 8.70±0.04 g and 9.62±0.05 cm, respectively. The mortality rate (M) in this group was 0.95±0.1 % and 12.86±0.1 % in the Group 1. The difference among the W and L values, specific growth rates (SGR) and M averages obtained from the groups was found to be statistically significant (P

Bu araştırmada has kefal balığının (Mugil cephalus) yumurtalarından havyar elde etmek amacıyla toplam 13 adet kefalden, kuru tuzlama işleme tekniği için 7 adet balıktan alınan yumurtalar, etrafını saran zarlarından ayıklandıktan, kılcal damar ve kanları temizlendikten sonra ağırlığı 280 g tespit edilen yumurtalar alınmıştır. Aynı şekilde ham ürün için 6 adet balıktan 243 g yumurta elde edilmiştir.Kuru tuzlama işleme tekniği sonucunda elde edilen ürünün ve karşılaştırma yapmak amacıyla işlenmemiş ham ürünün oda sıcaklığında ve laboratuar koşullarında kimyasal olarak; ham kül, ham nem, ham yağ, ham protein ve tuz analizleri, mikrobiyolojik olarak; total aerob mezofil bakteri, Staphlyococcus micrococcus ve koliform bakteri sayıları ve duyusal olarak; renk, koku, tat ve tuzluluk özellikleri incelenmiştir.Kimyasal bulgular ham üründe %61,8 nem, %23,9 protein, %9,9 yağ, % 5,1 kül ve % 3 tuzluluk ve kuru tuzlama işleme tekniğiyle işlenmiş havyar ise; %3,9 nem, %24,5 protein, %11,5 yağ, % 9,5 kül ve %8.7 tuz içeriğine sahiptir. Mikrobiyolojik olarak; ham havyarda toplam mezofil aerob bakteri 7,5 x 106 işlenmiş havyarda 1,5 x 103 koliform bakteri ham ürün 8,5 x 104 işlenmiş ürün 1,3 x 103 ve Staphlyococcus micrococcus ham ve tuzlanmış havyarda ?1.0 x 101 olarak bulunmuştur. Yapılan bu çalışmada ürünün duyusal olarak değerlendirilmesi sonucu, renk mükemmel, koku ve tadı iyi ve tuzluluk yönüyle de orta derecede bulunmuştur.Anahtar Kelimeler: Mugil cephalus, has kefal, kuru tuzlama işleme tekniği, havyar. For this study, the total of 13 mugil fishes (Mugil cephalus) was used for get caviar;, caviar of 7 fishes was tried with dry salt method; eggs tried made up 280 g with removed membrane, blood and capillars. Also 6 fishes were used for raw material and contained 243 g of caviar.The product originated by means of dry salting method and the raw product was analyzed in laboratory conditions for chemical contents such as follows:, moisture, protein, fat, ash and salt, and microbiological contents such as follows: total mesophilic aerobic bacteria, coliform group bacteria and Staphlyococcus/Micrococcus. Dry salted product was sensorically examined for colour, smell, flavour and taste of salting.Chemical results of raw product for moisture, protein, fat, ash and salt contents was %61.8 , %23.9 , %9.9 , % 5.1 , % 3 respectively, and chemical analysis of dry salted caviar was also %3,9 , %24,5 , %11,5 , % 9,5 , %8.7 respectively. Microbiologically, it was determined that mesophilic aerobic bacteria, and coliform group bacteria for both raw and dry salted caviar as 7,5 x 106 , 1,5 x 103 , 8,5 x 104 , 1,3 x 103 respectively. But, Staphlyococcus/ Micrococcus was not growth. In addition, sensorically; colour, smell, flavour and taste of salting was determined as excellent, good and moderate.Key Words: Mugil Cephalus, mullet, dry salt method, caviar. 32

12th International Symposium on Pollutant Responses in Marine Organisms (PRIMO 12) -- MAY 09-13, 2003 -- Safety Harbor, FL WOS:000258845700009 PubMed: 18752317 In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M, of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time. (C) 2008 Wiley Periodicals, Inc. Natl Inst Environm Hlth Sci, Int Soc Study Xenobiot, Univ Florida, Coll Pharmacy, Biosense Labs, Mol Light Technol Res, Hamilton Thorne Biosci, Elsevier Publishers, US Geol Survey, BRD FL Integrated Sci Ctr

31st Congress of the Federation-of-European-Biochemical-Societies (FEBS) -- JUN 24-29, 2006 -- Istanbul, TURKEY WOS:000246548200011 PubMed: 17113746 Information on the mechanism of metal ion inhibition of NADPH-cytochrome P450 reductase is limited. The purpose of the present paper was to elucidate in vitro effect of Hg(+2), Cd(+2), Ni(+2), Cr(+3) and Zn(+2) ions on the purified mullet NADPH-cytochrome P450 reductase. NADPH-cytochrome P450 reductase was purified from detergent-solubilized liver microsomes from leaping mullet (Liza saliens). All of the metal ions caused inhibition of the enzyme activity except Zn(+2). At 50 mu M metal concentration, Hg(+2) inhibited the cytochrome P450 reductase activity completely (100%), while, at the same concentrations, Cd(+2), Cr(+3) and Ni(+2) caused 66%, 65% and 37% inhibition, respectively. At 50 mu M metal concentration, Zn(+2) had no apparent effect on cytochrome P450 reductase activity. The IC(50) values of HgCl(2), CrCl(3), CdCl(2) and NiCl(2) were estimated to be 0.07 mu M, 24 mu M, 33 mu M and 143 mu M, respectively. Of the metal ions tested, Hg(+2) exhibited much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than the others. All four metal ions displayed noncompetitive type of inhibition mechanism for the purified reductase as analyzed by Dixon plot. K(i) values of Hg(+2), Cr(+3), Cd(+2) and Ni(+2) were calculated from Dixon plots as 0.048 mu M, 18 mu M, 73 mu M and 329 mu M, respectively. (c) 2006 Elsevier Ltd. All rights reserved. Federat European Biochem Soc

In this study, feral leaping mullet (Liza saliens) liver microsomal 7-ethoxyresorufin O-deethylase (EROD), and cytosolic glutathione S-transferases (GSTs) activities were investigated using 7-ethoxyresorufin, 1-chloro-2,4-dinitrobenzene (CDNB), and ethacrynic acid (EA) as substrates, respectively. The average EROD activity was found as 1139±175 pmol resorufin/min/mg protein. The average GST activities towards CDNB and EA were found as 1364±41 and 140±19 nmol/min/mg protein, respectively. We have, then, investigated the in vitro effects of some metals and detergents on CYP1A and GST activities in leaping mullet liver. Leaping mullet liver microsomal EROD activity was significantly inhibited by Hg (0.1 mM), Ni (0.1 mM), Cd (0.1 mM), Cu (0.1 mM), Zn (0.1 mM), Sb (0.1 mM), Fe2+ (1 mM), Co (1 mM), Al (1 mM), and Fe3+ (1 mM), with the percent inhibition of 80, 80, 77, 75, 70, 69, 56, 53, 46, and 44, respectively. Similarly, conjugation of CDNB catalyzed by GST was inhibited significantly to lesser extend by Hg (0.1 mM), Sb (0.1 mM), Cd (0.1 mM), Cu (0.1 mM), Zn (0.1 mM), Fe3+ (1 mM), Co (1 mM), and Fe2+ (1 mM), with the percent inhibition of 70, 69, 65, 61, 54, 51, 47, and 43, respectively. The degrees of inhibition observed on GST catalyzed EA conjugation by Hg (0.1 mM), Cd (0.1 mM), Sb (0.1 mM), Cu (0.1 mM), and Zn (0.1 mM) were 86, 78, 69, 51, and 42, respectively. In addition to metals, the effect of various detergents on leaping mullet liver EROD, GST-CDNB, and GST-EA activities were studied. It was found that ionic detergents strongly inhibited the EROD activity, whereas much less inhibitions were observed with GST catalyzed activities. Therefore, the CYP1A inhibition potencies of metals and detergents suggest that their contribution to the overall CYP1A induction in polycyclic aromatic hydrocarbons contaminated environmental samples has to be taken into account for better interpretation of environmental studies. © 2006 Elsevier Inc. All rights reserved.

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.

This study is the first detailed work on the age and growth of the leaping mullet (Liza saliens, Risso 1810) in the central Mediterranean. During the period 1991-1995 the age and growth of leaping mullet from the Messolonghi -Etoliko lagoon system (western Greek coast) were studied. Age and growth determinations were based upon otolith samples taken from 537 fish. Marginal increment analysis was used to validate age determination. Annulus formation took place around November each year. The back-calculated lengths at age estimated from the otoliths showed no differences between sub-areas of the lagoon system and the recorded limited between-years variability showed no persistent temporal pattern. The maximum age of leaping mullet in the Messolonghi - Etoliko lagoon was 5 years for males and 6 years for females. The von Bertalanffy equation (L‡=32.99±1.25 cm, k=0.258 ±0.017 year-1, t0=-0.47±0.04 year) accurately describes the growth of the total length of leaping grey mullet for all life stages (fry, juveniles and adults). A large spread and length overlap characterized the age groups. The estimated Length-Weight relationships were common for the two sexes (W=0.0079L3.01).

oz DOĞAL KEFAL BALIĞI (MARVAKİ) KARACİĞERİNDEN SİTOKROM P4502B-TİPİ PROTEİN: SAFLAŞTIRILMASI, BİYOKİMYASAL VE İMMÜNOLOJİK KARAKTERİZASYONU BOZCAARMUTLU, Azra Doktora, Biyokimya Tez Yöneticisi: Prof. Dr. Emel ARINÇ Ekim 2002, 209 sayfa Sitokrom P450'ler endojen ve ekzojen maddelerin oksidatif metabolizmasında önemli bir rol oynayan ve çok sayıda biyokimyasal reaksiyonda katalizör görevi gören hemoprotein süper ailesini oluşturur. Farklı hayvan gruplarında bu çeşitli reaksiyonların miktarı ve fonksiyonu büyük oranda farklı P450 proteinlerinin katılımına, katalitik fonksiyonuna ve regülasyonüna bağlıdır. Sitokrom P4502B alt ailesi genelde deney hayvanlarında ksenobiyotiklerin detoksifikasyonu ile ilgilidir ve ayrıca hormonları, testosteron ve androstediyonlan ve önemli pharmakolojik kimyasalları metabolize eder. Bu önemli rolü birçok biyokimyacı, pharmakolog ve toksikolog tarafından fark edilmiş ve birçok farklı memelide çalışılmıştır. Benzer genler balıkda da çok sayıda bulunabilir.Bu çalışmada, sitokrom P4502B4 homologuna karşı üretilen antikor ile kuvetli reaksiyon veren ve birinci iyon değişim kolonundan elde edilen bazik sitokrom P450 elektroforetik homojenlikte iki iyon değişim kolon kromatografisi (birinci ve ikinci DEAE-selüloz) ve üç farklı bir birini takip eden adsorpsiyon (Hidroksilapatit) kolon kromatografisi ile saflaştınldı. Saflaştınlan sitokrom P450 spektral, elektroforetik, biyokatalitik ve immunokimyasal özelliklerine göre karakterize edildi. Saflaştırılan sitokrom P450 SDS-poliakrilamit jel elektroforezin de moleküler ağırlığı 49300 olan tek bant verdi. Saf sitokrom P450, mutlak spektrumu 417 nm'de, ditiyonit ile indirgenmiş CO-fark spectrumu 450 ran dalga boyunda maksimum absorbans verdi. Saflaştınlan sitokrom P450 sentetik lipit ve kefal balığı karaciğerinden saflaştırılan P450 redüktaz enzimi ile yeniden oluşturulan mikrozomal monooksijenaz sisteminde benzfetamin, kokain, eritromisin ve etilmorfin N-demetilasyonunda ve pentoksirezorufinin O- depentilasyonunda aktif olduğu bulundu. Fakat, diğer monooksijenaz izozimlerinin substratlanm (anilin, laurik asit, 7-etoksirezorufın, 7- metoksirezorufin ve 7-benziloksirezorufin gibi) metabolize etmedi. Benzfetamin N-demetilaz activitesi yeniden oluşturulmuş ortamda bir sitokrom P4502B inhibitörü olan proadifen ile inhibisyona uğradı. Bunlara ek olarak, saflaştırılan sitokrom P450, sitokrom P4502B4 homologuna karşı üretilen antikor ile reaksiyon verdi. Yukarıdaki çalışmalar gerçekleştirildikten sonra elde edilen sonuçlar kefal karaciğerinden saflaştırılan sitokrom P450'nin bir sitokrom P4502B-tipi protein olduğunu gösterdi. Bu çalışmada, ilk defa sitokrom P4502B alt ailesinin bir üyesi balıktan saflaştırıldı ve karakterize edildi. Anahtar kelimeler: Sitokrom P4502B-tipi protein, saflaştırma, karakterizasyon, kefal (Uza saliens), monooksijenazlar, proadifen, benzfetamin, kokain, eritromisin, etilmorfin, 7-pentoksirezorufin vı ABSTRACT CYTOCHROME P4502B-TYPE PROTEIN FROM FERAL LEAPING MULLET {LIZA SALIENS) LIVER MICROSOMES: ITS ISOLATION AND BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION BOZCAARMUTLU, Azra Ph. D., Department of Biochemistry Supervisor: Prof. Dr. Emel ARINÇ October 2002, 209 pages Cytochrome P450s constitute a superfamily of hemoproteins that play important roles in oxidative metabolism of endogenous and exogenous compounds, and serve as catalysts that are significant in numerous and diverse biological pathways. The extent to which these various pathways or functions occur in different animal groups depend on a large degree on the participation of different P450 proteins present, their catalytic function and their regulation. Cytochrome P4502B subfamily is generally associated with detoxifying metabolism of xenobiotics in experimental animal species and also metabolizes hormones, testosterone and androstedione and several important pharmaceutical agents. The important role of cytochrome P4502B subfamily has been recognized by many biochemists, pharmacologists and toxicologist and has been studied in many different mammalian species. There could be a large number of homologous genes in fish. inIn this study, basic cytochrome P450 that cross-reacted with antibody produced against cytochrome P4502B4 homologue, eluted from the first DEAE- cellulose column was purified to electrophoretic homogeneity by using first and second anion exchange chromatography, and three successive hydroxylapatite column chromatographies. Purified cytochrome P450 was characterized with respect to spectral, electrophoretic, immunochemical and biocatalytic properties. Purified cytochrome P450 gave a single band on SDS-polyacrylamide gel electrophoresis having monomer molecular weight of 49300 Da. Absolute absorption spectrum of the purified cytochrome P450 showed maximum at 417 ran and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. Purified cytochrome P450 was found to be active in the N- demethylation of benzphetamine, cocaine, erythromycin and ethylmorphine and O-depenthylation of pentoxyresorufin in the reconstituted systems containing purified cytochrome P450 reductase from leaping mullet and synthetic lipid. However, it was unable to catalyze the oxidation of the other monoxygenase substrates such as aniline, lauric acid, 7-ethoxyresorufin, 7-methoxyresorufin and 7-benzyloxyresorufin. Benzphetamine N-demethylase activity was inhibited with proadifen, a cytochrome P4502B inhibitor in reconstituted system. In addition, purified cytochrome P450 showed strong cross-reactivity with the antibody produced against sheep lung cytochrome P4502B4 homologue. The results obtained after accomplishing above studies, strongly suggested that cytochrome P450 purified from leaping mullet is a cytochrome P4502B-type protein. In this study, for the first time, a member of cytochrome P4502B subfamily was purified and characterized in fish species. Key words: Cytochrome P4502B-type protein, purification, characterization, leaping mullet (Liza saliens), monooxygenases, proadifen, benzphetamine, cocaine, erythromycin, ethylmorphine, 7-pentoxyresorufin IV 235

In this study, we examined whether levels of P4501A mRNA expression were naturally induced in feral fish, Liza saliens , and whether CYP1A protein levels and associated enzyme activity, EROD, were also increased. Induction of mRNA was measured using a nucleic acid hybridization technique. For the hybridization studies, a new 33-mer oligonucleotide probe 5′-dCTC ATC CAG CTT CCT GTC CTC GCA GTG ATC AAT-3′ was designed, which corresponded to the totally conserved amino acid motif of CYP1A protein from positions 291 to 301 among the various fish species. Results of Northern blot analysis revealed that RNA isolated from the liver of mullet collected from the highly contaminated region of Izmir Bay with a dissolved and dispersed petroleum hydrocarbon content of 12.45 μg l −1 gave a strong hybridization signal, whereas only a weak hybridization signal was detected in the liver RNA of fish caught from the reference site containing less than 1 μg l −1 of petroleum hydrocarbons. Similarly, fish from the contaminated site had approximately 80 times more EROD activity than the feral fish captured from the reference site. Studies using polyclonal antibodies produced against purified mullet CYP1A also showed the similar trend. In conclusion, feral leaping mullet caught from contaminated water displayed induction of CYP1A at three levels of expression, namely, mRNA, apoprotein and catalytic activity.

A 2,037 bp CYP1A1 cDNA (GenBank AF072899) was cloned through screening of a lambda ZipLox cDNA library constructed from the liver of a leaping mullet (Liza saliens) fish captured from Izmir Bay on the Aegean coast of Turkey using rainbow trout CYP1A1 cDNA as a probe. This clone has a 130 bp 5'-flanking region, a 1,563 bp open reading frame (ORF) encoding a 521-amino acid protein (58,972 Da), and a 344 bp 3'-untranslated region without a poly (A) tail. Alignment of the deduced amino acids of CYP1A1 cDNAs showed 58% and 69-96% identities with human and 12 other fish species, respectively. Southern blot analysis suggested that this CYP1A1 cDNA was from a single-copy gene. Based on the comparison with CYP1A1 genes reported for fish and mammals, the leaping mullet CYP1A1 gene is probably split into 7 exons. The intron insertion sites were predicted. Alignment of the CYP1A1 cDNA encoded amino acids from 13 fish and 7 mammalian species disclosed differences in highly conserved amino acids between aquatic and land vertebrates. The possible associated secondary structure; conserved motifs and substrate-binding sites were discussed. The phylogenetic relationships of CYP1A1s among 13 fish species were analyzed by a distance method. (C) 2001 John Wiley & Sons, Inc. J Biochem. Mol Toxicol 15:243-255, 2001.

IV ÖZET İZMİR VE ALİAĞA KÖRFEZİNDE MEVSİMSEL OLARAK AVLANAN BAZI EKONOMİK BALIK TÜRLERİNDE ORGANİK KLORLU PESTİSİT KAUNTDLARININ ARAŞTIRILMASI ULUOCAK, H. Belgin Doktora Tezi Su Ürünleri Temel Bilimler Ana Bilim Dalı Tez Yöneticisi ; Prof. Dr. Özdemir EGEMEN Mayıs 2000, 78 Sayfa Bu çalışmada Aliağa, Foça, Tuzla (Homa), Urla ve Mordoğan'dan dört tur balık ( kefal, barbun, çipura, dil) mevsimsel olarak toplanmış ve analize alınmıştır. Balık dokusunda OC'lu pestisitler GC-ECD ile analiz edilmiş ve GC - MS ile doğrulanmıştır. Elde edilen bulgulara göre tüm numunelerde OC pestisitlerden DDT'nin metaboliti olan DDE bulunmuştur.DDE daha karalı bir bileşik olup, DDT'nin eskiden kullanılmış olduğunu göstermektedir.Anahtar Kelimden ec-BHC, 0-BHC,5-BHC,A-BHC, Heptaklor Aldrin, Heptaklorexoepoksit, Heptaklorendoepoksit, Dieldrin, DDE Endrin, TDE (DDD), DDT ve Metoksiklor kalıntısı; balık kas dokusu ; GC-ECD. VI ABSTRACT THE STUDY OF THE RESIDUE ANALYSIS OF ORGANOCHLORIDE PESTICIDES IN SEVERAL KINDS OF FISH WHICH ARE CONSUMABLE FROM ALIAGA AND IZMIR BAY IN FOUR SEASONS ULUOCAK, H. Belgin PhJDSu Ürünleri Temel Bilimler A.B.D. Supervisor: Prof. Dr. Özdemir EGEMEN May 2000, 78 pages In this study four kinds offish ( red mullet, grey mullet, seabream and sole ) from Aliağa, Foça, Tuzla (Homa), Urla and Mordoğan were analysed. The chlorinated pesticides were analysed in fish tissue by GC-ECD and confirmed by GC - MS. DDE had been seen in all of the samples. DDE is the main metabolite of DDT. DDE is a more stable compound. So we can say that DDT was used as pesticide.VII Keywords: a-BHC, p-BHC, 8-BHC, A-BHC, Heptachlor, Aldrin, Heptachlorexoepoksit, Heptachlorendoepoksit, Dieldrin, DDE, Endrin, TDE (DDD), DDT and Metoxychlor. 77

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7- methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7- methoxyresorufin O-demethylase (MROD) activity were studied. 7- ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by ?- naphthoflavone (ANF). At 0.5 and 2.5 µM, ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1. (C) 2000 Elsevier Science Inc.

ÖZET Yüksek Lisans Tezi KEFAL (Mugil sp.) YAVRULARININ BESLENMESİNDE FARKLI ORANLARDA KARMA YEM VE BALIK ETİ KULLANIMININ BÜYÜME PERFORMANSINA ETKİLERİ Yusuf BO/.KURT Ankara Üniversitesi Fen Bilimleri Enstitüsü Su Ürünleri Anabilinı Dalı Danışman : Prof. Dr. Selçuk SEÇER Bu çalışmada, doğadan yakalanan Mugil sn. yavrularının beslenmelerinde %32 ham protein içeren azan pelet yemine %25, %50, %75 ve %100'ü oranlarında karıştırılan pul balığının (Leiognathus Junzingeri S.1898) kıs aylarındaki büyüme performansına olan etkileri araştırılmıştır. Deneme başlangıcında ortalama ağırlığı 3.4611 ± 0.323 g ve ortalama total boyu 7.484 i 0.157 cm ilan Mugil sp. yavruları, 2 tckcrrürlû 5 grup (%2S, %5U, %75, %1UU ve kontrol) olarak planlanan leneme sonunda sırasıyla 5.011 ± 11.118 g, 4.814 ± 0.343 g, 4.425 ± 11.244 g. 4.0011 1 0.282 g ve 4.J00 + ).141g canlı ağırlığa ve 7.497 ± 0.004 cm. 7.419 ± 0.285 cm, 7.825 ± 0.318 cm, 7.903 ± 0.033 cm ve 7.775 + M90 cm total uzunluğa erişmişlerdir. Üzerinde durulan parametreler bakımından deneme grupları içerisinde en iyi değerler, canlı ağırlık kazancı bakımından, 1.211 ± 0.016 g ile pelet yeme %25 oranında pul balığı {Leiognathus klunzingeri S.1898) ilave edilen grupta, günlük canlı ağırlık kazancı 0.013 ± 0.045 g ile kontrol grubunda, spesifik ağırlıkça büyüme oranı %0.363 + 0.037 ile kontrol grubunda, spesifik boyca büyüme oranı %0.84 ± 0.011 ile pelet yeme %50 oranında pul balığı {Leiognathus klunzingeri S.1898) ilave edilen grupta, yem dönüşüm etkinliği 3.429 ± 0.000 ile kontrol grubunda, kondisyon faktörü 1.150 ± 0.212 ile pelet yeme %25 ve %50 oranında pul balığı (Leiognathus klunzingeri S.1898) ilave edilen grupta ve en yüksek yaşama oranı ise %95 i 7.070 ile sadece pul balığı ile yemleme yapılan grupta saptanmıştır. 2000.41 sayfa ANAHTAR KELİMELER : Kefal, Chelon (Mugil) labrosus. Yaş yem, Yetiştiricilik ABSTRACT Master Thesis THE EFFECTS OF USING DIFFERENT RATES OF ARTIFICIAL FEED AND FISH MEAT ON GROWTH PERFORMANCE OF GREY MULLET FRIES Yusuf BOZKURT Ankara University Graduate School of Natural and Applied Sciences Department of Fisheries and Aquaculturc Supervisor : Prof. Dr. Selçuk SEÇER In this study, the effects of Leiognathus klunzingeri S. 1898, that were mixed to the pellet feed :ontains 32 % crude protein) at different ratios as 25 %, 50 %,75 % and 100 % on the growth crformancc of Mugil sp. fry captured from the nature were investigated. At the begining of the experiment, the mean weight and length were measured as 3.460 ± 0.323g and.484 ± 0.157 cm respectively. At the end of the trial that was planned as two dublicatc of five groups (25 'o, 50 %, 75 %,100 % and control), the final mean weights and lengths were found as follows; 5.011 ± i.l 18 g, 4.814 ± 0.343 g, 4.425 ± 0.244 g, 4.000 ± 0.282 g, 4.300 ± 0.141 g and 7.497 ± 0.004 cm, 7.419 ±.285 cm, 7.825 ± 0.318 cm, 7.903 ± 0.033 cm and 7.775 ± 0.190 cm respectively. Among the treatment groups the highest mean growth rate and mean daily weight gain were tutaincd at the group A (fish meat level is 25 %) and group E (control) respectively. The highest spesific »rowth rate as weight and length were observed as 0.363 ± 0.037 %/day and 0.840 ± 0.01 1 %/day at the :ontrol group and group B (fish meat level is 50 %) respectively. Feed conversion efficiency was found idler than the others at the control group with the value of 3.429 ± 0.000. Condition factor was determined as the highest 1.150 ± 0.070 and 1.150 ± 0.212 at the group A (fish meat level is 25 %) and group B (fish meat level is 50 %). At the end of the trial the highest survival rate was observed at the group D (fish meat level is 100 %) with percentage of 95 ± 7.070 %. 2000,41 pages. Key Words : Mullet, Chelan (Mugil) labrosus. Moist feed. Breeding 41

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demethylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by cl-naphthoflavone (ANF). At 0.5 and 2.5 mu M, ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine. N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1. (C) 2000 Elsevier Science Inc. All rights reserved.

Cytochrome P4501A1 was purified to electrophoretic homogeneity from the liver microsomes of feral fish leaping mullet (Liza saliens) collected in Izmir Bay, Aegean coast of Turkey. Purification of cytochrome P4501A1 involved anion exchange chromatography of Emulgen 913-cholate solubilized microsomes on first- and second-DEAE-cellulose columns, hydrophobic interaction chromatographies of the partially purified cytochrome P4501A1 on Porapak Q and phenyl-Sepharose CL-4B and further purification on adsorption chromatography on the hydroxylapatite column. Finally, it is further concentrated and purified on the third DEAE-cellulose column. The purified cytochrome P4501A1 was characterized with respect to spectral, electrophoretic, immunochemical and biocatalytic properties. Cytochrome P4501A1, purified 32-fold with a specific content of 15-17 nmoles P450 (mg protein)-1, produced a single band on SDS-polyacrylamide gel electrophoresis having monomer molecular weight of 58,000 +/- 500. Absolute absorption spectrum of the purified cytochrome P4501A1 fractions showed maximal absorption at 417.5 nm and CO-difference spectrum of dithionite-reduced cytochrome P4501A1 gave a peak at 448 nm. Purified P4501A1 was found to be active in the O-deethylation of 7-ethoxyresorufin in the reconstituted system containing purified fish liver cytochrome P450 reductase and synthetic lipid. However, it was unable to catalyze the oxidation of the other monooxygenase substrates such as benzphetamine and aniline known to be specific for the other isozymes. Purified L. saliens liver microsomal cytochrome P4501A1 showed strong cross-reactivity with the antibodies directed against the cytochrome P4501A1 homologues purified from other teleost species such as rainbow trout and scup. Spectral, electrophoretic, immunochemical and biocatalytic properties of the purified cytochrome P4501A1 strongly suggested that it is the CYP1A1 in the L. saliens liver.

ÖZET KÖYCEĞİZ DALYANI'NDA KEFALLERİN (Liza aurata RISSO, 1810 ve Mugil cephalus LINNAEUS. 1758) BÜYÜME ORAN VE İLİŞKİLERİ Bu çalışmada, Mart 1994-Şubat 1995 tarihleri arasındaki dönemde Köyceğiz Dalyanı'nda yaşayan Liza aurata (RISSO, 1810) ve Mugil cephalus (LINNAEUS, 1758) türlerinin eşey oranlan, yaş kompozisyonları, yaş-boy, yaş-ağırlık, boy-ağırlık ilişkisi ve kondisyon faktörü gibi büyüme oran ve ilişkileri saptanmıştır. Bu çalışmada yaş gruplarına göre ortalama total boy ve ağırlıklara bakılarak dişi bireylerin erkek bireylere nazaran boy ve ağırlıkça daha iyi büyüdükleri tesbit edilmiştir. Ayrıca boy-ağırlık arasındaki ilişkiyi gösteren katsayının (r) l'e yakın çıkması boy-ağırlık arasında iyi bir ilişkinin olduğunu göstermektedir. Yine kondisyon faktörü değerlerinin l'e yakın çıkması ortam ile Liza aurata (RISSO, 1810) ve Mugil cephalus (LINNAEUS, 1758) bireyleri arasında yeterli diyebileceğimiz bir beslenme dengesinin varlığını göstermektedir. VIII SUMMARY GROWTH RATE AND GROWTH RELATIONSHIPS OF THE MULLETS {Liza aıırata RISSO, 1810 and Mugil cephalus LINNAEUS, 1758) FROM KOYCEGIZ DALIAN In this study, which was carried out between March 1994 and February 1995, the age-length, the age-weight, the length-weight relationship, the age composition, the sex ratios, and the condition factors of Liza aurata (RISSO, 1810) and Mugil cephalus (LINNAEUS, 1758) in the Dalian of Köyceğiz were investigated. The female individuals showed a more rapid growth in length and in weight than male individuals. The factor (r) showed a value close to 1 indicating a fairly close relationship in the length-weight of these two species. The value of the found condition factors suggests a suitable relationship of feeding between the aquatic ecosystem of the Köyceğiz Dalian and Liza aurata (RISSO, 1810) and Mugil cephalus (LINNAEUS, 1758). IX 60

oz KEFAL (LISA SALIENS) KARACİĞERİNDEN GLUKOZ-6-FOSFAT DEHİDROGENAZ ENZİMİNİN İZOLASYONU VE KARAKTERİZASYONU Elay, Yurdaer Yüksek Lisans, Biyokimya Bölümü Tez Yöneticisi: Prof. Dr. Emel Arınç Eylül 1999, 103 sayfa Bu çalışmada pentoz fosfat yolunun ilk enzimi olan glukoz-6-fosfat dehidrogenaz, İzmir Körfezi'nden toplanan kefal {Lisa saliens) balığının karaciğerinden izole edildi ve çeşitli biyokimyasal özellikleri karakterize edildi. Enzimin izolasyonu amonyum sülfat çöktürmesi ve 2',5'-ADP- Sepharose 4B afinite kromatografisi yöntemleri ile yapıldı. SDS- poliakrilamit jel elektroforezinde enzim tek bir protein bandı verdi veenzimin monomer moleküller ağırlığı 64000 Dalton olarak hesaplandı. İlki 7.0-8.0 değerleri arasında ve ikincisi 9.5'ten 10.5'e doğru artmak suretiyle, yüksek enzim aktivitesinin gözlendiği iki farklı pH aralığı saptandı. Enzimin doğal substratı glukoz-6-fosfat için Vmax ve Km değerleri sırasıyla 244 nmol/dak/ml ve 0.098 mM olarak hesaplandı. Enzimin spesifik koenzimi NADP+ için Vmax ve Km değerleri sırasıyla 256 nmol/dak/ml ve 0.008 mM olarak hesaplandı. Enzim kinetiğinin hem glukoz-6-fosfat için hem de NADP+ için aynı olduğu ve Michaelis-Menten kinetiğine uyduğu görüldü. Magnezyum iyonunun, çok düşük derişimlerde dahi enzimin aktivitesini stimule ettiği görülürken, kadmiyum ve nikel iyonlarının da enzim aktivitesini artırdıkları gözlendi. Çinko iyonunun enzimi inhibe ettiği görüldü. Alüminyum iyonunun enzim aktivitesi üzerinde önemli bir etkisinin olmadığı görüldü. Gliserolün varlığında enzimin ısıya maruz kaldığında stabilitesini koruduğu gözlendi. Anahtar kelimeler: Pentoz fosfat yolu, glukoz-6-fosfat dehidrogenaz, karakterizasyon, kefal (Lisa saliens) vı ABSTRACT MULLET {LISA SALIENS) LIVER GLUCOSE-6-PHOSPHATE DEHYDROGENASE; ISOLATION AND CHARACTERIZATION Elay, Yurdaer M. S., Department of Biochemistry Supervisor: Prof. Dr. Emel Arınç September 1999, 103 pages In this study, glucose-6-phosphate dehydrogenase, the first enzyme of the pentose phosphate pathway was isolated from the liver of leaping mullet (Lisa saliens) collected in Izmir Bay, Agean coast of Turkey and several biochemical properties of the enzyme was characterized. Isolation of the enzyme involved ammonium sulfate precipitation and 2',5'-ADP-Sepharose 4B affinity chromatography methods. The enzyme showed a single protein band on SDS-polyacrylamide gel iii 103

IV ÖZET AĞ KAFESLERDE KEFAL ( Chelon labrosus R. 1826 ) YETİŞTİRİCİLİĞİ ALTUNOK, Muhammet Yüksek Lisans Tezi, Su Ürünleri Yetiştiriciliği Bölümü Tez Yöneticisi: Doç. Dr. Osman ÖZDEN Ocak 1998, 62 sayfa Bu çalışmada kalın dudaklı kefal yavrularının denizdeki ağ kafeslere uyumu ve büyütülmesi amaçlanmıştır. Doğadan toplanan yavrular aynı stok yoğunluğunda 4 farklı grupta stoklanarak 7 ay boyunca, farklı ham protein oranlarına sahip yemlerle beslenmişlerdir. Yavrular 6 m3 Kik ağ havuzlara 1 6 adet/m3 olarak stoklanmışlardır. Çalışmada, hazırlanan % 25, 30 ve 35' lik ham proteinli karmalar ile ticari sazan yemi kullanılmıştır. Grupların başlangıç ağırlıkları ortalama 6.2 ± 0.93 g, sonuçta A grubu 51. 1± 7.06 g, B grubu 82.5 ± 6.04 g, C grubu 77.1 ± 5.78 g ve D grubu 58.4 ± 3.05 g olmuşlardır. Bütün grupların yaşama oranları % 95' in üzerinde gerçekleşmiştir. Anahtar kelimeler: Chelon labrosus, ağ kafes kültürü, adaptasyon, gelişme, pelet yem. V ABSTRACT CAGE CULTURE OF THICK LIPPED MULLET (C labrosus R. 1826) ALTUNOK, Muhammet Msc in Fisheries Faculty Supervisor. Doç. Dr. Osman Özden January 1998, 62 pages The purpose of this work was to obtain some preliminary data on acclimation of fingerlings and grow-out of Chelon labrosus under intensive conditions in sea netcages. Fingerlings of C. labrosus collected from the sea shore and stocked in cages at same stocking densities were reared for 7 months with suplementary feeding. Four 6 m3 net in a cage were used for the experiments. Stocking densities were 16 mullet / m3 in netcages, respectively. Three diets containing 25, 30 and 35% protein levels were prepared. In last group was fed with commercial carp feed. Initial mullet weight was 6.2 ± 0.93 g. Final mean weights of mullets were group A 51.1 ± 7.06 g, group B 82.5 ± 6.04 g, group C 77.1 ± 5.78 g and 58.4 ± 3.05 g for group D in 210 days. Mullets reached a maximum weight with 82.5 g in B group. Optimum fish performance was achieved at 30% dietary protein. Survival was, in all groups over of 95% after 210 days in seacages. Key words: Chelon labrosus, seacage culture, acclimation, growth, pellet feeding 62

oz NADH-SITOKROM b5 REDUKTAZ ENZİMİNİN KEFAL KARACİĞER MİKROZOMLAREVDAN KISMİ OLARAK SAFLAŞTIRILMASI VE KARAKTERİZASYONU KABAKLAR, Zahide Yüksek Lisans, Biyokimya Bölümü Tez Yöneticisi: Prof. Dr. Emel ARINÇ Ocak 1998, 108 sayfa NADH-sitokrom b5 redüktaz enzimi deterjan ile çözünürleştirilmiş kefal karaciğer mikrozomlarının birbiri ardından iki DEAE-sellüloz ve 5' ADP agaroz afinite kolon kromatografilerine uygulanması ile saflaştırıldı. Başlangıçtaki mikrozomlara oranla %10'luk bir verim ile 52.1 kez saflaştırılmış olarak elde edildi. Yaklaşık monomer moleküler ağırlık, SDS-poliakrilamit jel elektoforezi ile 33800 ± 1000 olarak bulundu. Ferrisiyanit elektron alıcısı olarak kullanıldığında, redüktaz pH 6.5 ve 7.5 arasında optimum aktiviteyi gösterdi. Sitokrom b5 substrat olarak kullanıldığında ise, maksimum enzim aktivitesi iki farklı pH da, 6.5 ve 8.5, elde edildi.Ferrisiyanit indirgenmesi için maksimum enzim aktivitesi 25 °C de gözlendi. Enzim, 37 °C de aktivitesinin %45 ini kaybetti. Ferrisiyanit için Km ve Vmax değerleri sırasıyla 50 uM ve 86.9 umol indirgenmiş ferrisiyanit/dak/mg olarak hesaplandı. Kofaktör NADH için Km ve Vmax değerleri doygunluk konsantrasyonunda ferrisiyanit varlığında, 45.4 uM ve 113.4 umol indirgenmiş ferrisiyanit/dak/ mg olarak bulundu. Kefal redüktaz enzimi tarafından sitokrom b5 indirgenmesinin Michaelis-Menten kinetiğine uymadığı gözlendi ve yaklaşık Km değeri 3 uM olarak bulundu. Sitokrom c indirgenme kinetiği sitokrom b5'in fonksiyonu olarak, sitokrom c ve sitokrom b5 redüktaz varlığında incelendi. Km ve Vmax değerleri sırasıyla 0.016 uM sitokrom b5 ve 26.6 umol sitokrom c/dak/mg enzim olarak bulundu. Sonuç olarak, balık karaciğer sitokrom b5 redüktaz enzimi memelilerden elde edilen karaciğer redüktazlanna bazı özellikler yönünden benzemekte iken, optimum sıcaklık ve sitokrom b5 kinetiği yönünden farklı özellikler göstermektedir. Anahtar kelimeler: Kefal, sitokrom b5 redüktaz, saflaştırma, karakterizasyon, balık karaciğeri, mikrozom, kinetik, sitokrom c indirgenmesi. vı ABSTRACT PARTIAL PURIFICATION AND CHARACTERIZATION OF NADH- CYTOCHROME b5 REDUCTASE FROM MULLET LIVER MICROSOMES KABAKLAR, Zahide M.S., Department of Biochemistry Supervisor : Prof. Dr. Emel ARINÇ January 1998, 108 pages NADH-cytochrome b5 reductase was partially purified from detergent solubilized mullet liver microsomes by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 52.1-fold and the yield was 10% with respect to microsomes. The apparent monomer Mr of reductase was determined to be 33800 ± 1000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed optimum activity between pH 6.5 and 7.5. The maximal activity of the enzyme with cytochrome b5 as a substrate was obtained at two different pH values, pH 6.5 and pH 8.5. The maximum activity of the enzyme for ferricyanide reduction was observed at 25 °C. Reductase lost 45% of its activity at 37 °C. K`,and Vnux values mof the enzyme for ferricyanide were calculated as 50 uM and 86.9 umol ferricyanide reduced/min/mg enzyme. The Km and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide was found to be 45.4 uM and 113.4 umol ferricyanide reduced/min/mg. On the other hand, cytochrome b5 reduction by mullet reductase did not obey Michaelis-Menten kinetics, and apparent Km for cytochrome b5, although not accurate, was determined as 3 uM. Kinetics of cytochrome c reduction as a function of cytochrome b5 in the presence of cytochrome c and cytochrome b5 reductase were investigated and the Km and Vmax values were found as 0.016 uM cytochrome b5 and 26.6 umol cytochrome c reduced/min/mg enzyme. The results suggest that some properties of fish liver cytochrome b5 reductase are similar to those of mammalian liver while optimum temperature and reduction of cytochrome b5 kinetics of fish enzyme are different from those of mammals. Keywords: Mullet, cytochrome b5 reductase, purification, characterization, fish liver, microsomes, kinetics, cytochrome c reduction. IV 108

Ill ÖZET Yüksek Lisans Tezi TATLI SU KEFALI ' NİN ( Leuciscus cephalus L.) DIŞ MORFOLOJİK ÖZELLİKLERİ VE OVARYUMLARININ HİSTOLOJİK YAPISININ ARAŞTIRILMASI Mesut URAL Fırat Üniversitesi Fen Bilimleri Enstitüsü Su Ürünleri Yetiştiriciliği Anabilim Dalı 1997, Sayfa: 40 Bu çalışmada 135 adet dişi Tatlı Su Kefali ' nin ( Leuciscus cephalus I,.) dış morfolojik özellikleri, ovaryumlarının makroskobik ve mikroskobik yapısı incelenmiştir. Kasım-1995, Kasım-1996 tarihleri arasında Keban Baraj Gölünden yakalanan Tatlı Su Kefallerinin morfometrik ölçümleri alındıktan sonra dişi balıkların ovaryumları çıkartıldı. Ovaryumlarında morfometrik ölçümleri alındı ve % 10 Nötral Formalin, Bouin' s ve Tamponlu Formalin solüsyonları içinde tesbit edildi. En iyi sonuçlar Bouin' s ve Tamponlu Formalin solüsyonlarından alındı. Tesbit edilen doku parçalarından parafin bloklar hazırlandı ve mikrotomda 5-7 mikronluk kesitler alınarak rutin incelemeler için Masson' un üçlü boyaması, P.A.S. boyası, Heamotoxylen -eozin boyamaları uygulandı. Balıkların, yaşlarının tesbiti için pul, otolit, operkulum ve vertebralarından yararlanıldı. Bu balıkların yumurtalarının süratli gelişiminin Şubat ayında başladığı (450 mikrometre) ve Ağustos ayında (650 mikrometre) sona erdiği gözlemlenmiştir. ANAHTAR KELİMELER: Tatlı su kefali, Morfoloji, Histoloji,Ovaryum,Keban Baraj gölü. IV SUMMARY Master Thesis A Study on External Morphology and Ovarium Histological of Fresh Water chubs (Leuciscus cephalus L) Mesut URAL Fırat University Graduate School of Natural and Applied Sciences Department of Fish Production and Breeding 1997, Page:40 In this study, external morphological characteristics, macroscobic and microscobic structure of ovaries of 135 female fresh water chubs ( Leuciscus cephalus L.) were examined. Specimens caught from Keban Dam Lake between November - 1 995, November - 1996, were measured macroscopically and then ovarium of female fishs were removed. Ovaries were measured macroscopically and fixed in % 10 Neutral formaline, Bouin's and Formaline with buffer solutions. The best results was obtained with Bouin's and Formaline with buffer. Fixed tissues were blocked by using paraffin and then blocked tissues were dissected in 5-7 Mm using microtome. Dissectted tissues were stained for rutine examinations by using Masson's threesome staining, P. A. S, Heamotoxylen and Eozin. At the some time, otolith, operculum and vertebra were examined in order to determine the ages of experimental fish. It is observed that the rapid growning of the eggs of these fishes starts at February (450 Mm) and ends at August (650 Mm) KEY WORDS: Fresh water chub, ovarium, histological, morphological, Keban Dam Lake. 40

öz SİTOKROM P4501A1 VE P450 REDÜKTAZIN DOĞAL KEFAL BALIĞI KARACİĞERİNDEN SAFLAŞTIRILMASI VE KARAKTERİZASYONU ŞEN, Alaattin Doktora, Biyokimya Tez Yöneticisi: Prof. Dr. Emel ARINÇ Ocak 1997, 151 sayfa Bu çalışmada, Izmir körfezinden toplanan doğal kefal (marvaki) balığının karaciğerinden sitokrom P4501A1 ve sitokrom P450 redüktaz homojen olarak saflaştınldı. Sitokrom P4501Al'in saflaştınlması, Emulgen 913 ve kolat ile çözünürleştirilmiş mikrozomlann iyon değişim kolon kromatografisi (birinci ve ikinci DEAE-selüloz), hidrofobik interaksiyon kolon kromatografisi, ( Porapak Q ve fenil-Sefaroz CL-4B) ve adsorpsiyon kolon kromatografisi (Hidroksilapatit) ile sağlandı. Saflaştınlan P4501A1 üçüncü DEAE-selüloz kolonu ile konsantre edildi. Sitokrom P4501A1 spektral, elektroforetik, biyokatalitik, ve immunokimyasal özellikleri incelenerek karakterize edildi. 32 Kez saflaştınlan ve özgül miktarı 15-17 nmol/mg protein olarak bulunan sitokrom P4501A1, SDS-poliakrilamit jel elektroforezin de moleküler ağırlığı 58000 ± 500 olan tek bir bant gösterdi. Mutlak spektrumu 417.5nm de, ditiyonit ile indirgenmiş CO-fark spektrumu 448nm dalga boylannda absorbans tepecikleri verdi. Saflaştınlan P4501A1 sentetik lipit ve balık karaciğer P450 redüktaz enzimi ile yeniden oluşturulan mikrozomal monooksijenaz sisteminde 7-etoksirezorufin O-deetilasyonunda aktif olmasına ragmen bilinen diğer monooksijenaz izozimlerinin substratlannı (benzfetamin ve anilin gibi) metabolizeetmedi. Kefal karaciğer mikrozomlanndan elde edilen sitokrom P4501A1 scup, alabalık ve cod gibi diğer teleost türlerinden saflaştırılan P4501A1 homologlarına karşı üretilen antikorlarla reaksiyon verdi. Spektral, elektroforetik, biyokatalitik, ve immunokimyasal özellikleri kefal karaciğerinden saflaştınlan sitokrom P450'nin P4501A1 izozimi olduğunu gösterdi. NADPH'ye bağlı sitokrom P450 redüktaz enzimi, 438-kez ve mikrozomlara oranla %17.5 verimle kefal karciğerinden saflaştınldı. Özgül aktivitesi 52.6 umol/dak/mg protein olarak saptandı. Enzimin moleküler ağırlığı SDS-poliakrilamit jel elektroforezinde 77000 ± 1000 olarak bulundu. Kefal karaciğer sitokrom P450 redüktaz enziminin mutlak spektrumu 378 ve 455nm dalga boylarında flavoproteinlere özgü olan iki tepecik gösterdi. Enzimin görünen Km değeri sitokrom c için 7.69 uM olarak saptandı ve enzimin 70 uM ve üzerindeki sitokrom c konsantrasyonlarında tamamen doymuş olduğu gözlendi. Bunlara ek olarak, enzimin biyokatalitik olarak aktif olduğu saptandı. Bütün bu sonuçlar saflaştınlan kefal karaciğer sitokrom P450 redüktaz'ın memeli P450 redüktazlanna spektral, elektroforetik ve biyokatalitik olarak benzer olduğunu gösterdi. Anahtar kelimeler: Sitokrom P4501A1, saflaştırma, karakterizasyon, kefal {Liza saliens), mavraki, monooksijenazlar, NADPH-sitokrom P450 redüktaz, 7-etoksirezorufin O-deetilaz (EROD). vı ABSTRACT PURIFICATION AND CHARACTERIZATION OF CYTOCHROME P4501A1 AND P450 REDUCTASE FROM FERAL LEAPING MULLET {LIZA SALIENS) LIVER MICROSOMES ŞEN, Alaattin Ph. D., Department of Biochemistry Supervisor: Prof. Dr. Emel ARINÇ January 1997, 151 pages In this study, cytochrome P4501A1 and cytochrome P450 reductase were purified to electrophoretic homogeneity from the liver microsomes of feral fish leaping mullet {Liza saliens) collected in İzmir Bay, Aegean coast of Turkey. Purification of cytochrome P4501A1 involved anion exchange chromatography of Emulgen 913-cholate solubilized microsomes on first- and second-DEAE-cellulose columns, hydrophobic interaction chromatographies of the partially purified cytochrome P4501A1 on Porapak Q and phenyl-Sepharose CL-4B and further purification on adsorption chromatography on the hydroxylapatite column. Finally, it is further concentrated and purified on third DEAE-cellulose column chromatography. It was characterized with respect to spectral, electrophoretic, immunochemical and biocatalytic properties. Cytochrome P4501 Al, purified 32-fold with a specific content of 15-17 nmoles P450 per mg protein, produced a single band on SDS-polyacrylamide gel electrophoresis having monomer molecular weight of 58000 ± 500. Absolute absorption spectrum of the purified cytochrome P4501A1 fractions showed maximal absorption at 417.5nm and CO- diff erence spectrum of dithionite-reduced cytochrome P4501A1 gave a peak at 448nm. Purified P4501A1 was found to be active in the O-deethylation of m7-ethoxyresorufin in the reconstituted system containing purified fish liver cytochrome P450 reductase and synthetic lipid. However, it was unable to catalyze the oxidation of the other monooxygenase substrates such as benzphetamine and aniline known to be specific for the other isozymes. Purified Liza saliens liver microsomal cytochrome P4501A1 showed strong cross-reactivity with the antibodies directed against the cytochrome P4501A1 homologues purified from other teleost species such as rainbow trout, scup and cod. Spectral, electrophoretic, immunochemical and biocatalytic properties of the purified cytochrome P4501A1 strongly suggested that it is the CYP1 Al in the Liza saliens liver. NADPH-cytochrome P450 reductase was purified 438-fold with a yield of 17.5% with respect to the initial amount present in the fish liver microsomes. The specific activity of the enzyme was found to be 52.6 umol cytochrome c reduced per minute per mg protein. The monomer molecular weight of the purified enzyme was calculated to be 77000 ± 1000 when subjected to electrophoresis on SDS-PAGE. The absorption spectrum of fish reductase showed two peaks at 378nm and 455nm. NADPH-dependent cytochrome c reductase activity of the purified Liza saliens liver cytochrome P450 reductase was found to be maximal when pH was between 7.4-7.8. The apparent Km of the purified enzyme was found to be 7.69 uM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer, pH 7.7 at room temperature and the enzyme was fully saturated by its substrate, cytochrome c, when the substrate concentration at or above the 70 uM. Furthermore, the purified enzyme was found to be biocatalytically active. These results suggested that the purified fish liver cytochrome P450 reductase is similar to its mammalian counterparts with respect to spectral, electrophoretic and biocatalytic properties. Key words: Cytochrome P4501A1, purification, characterization, leaping mullet (Liza saliens), monooxygenases, NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylase (EROD) IV 151

Bu çalışmada. Temmuz 1994 - Haziran 1995 tarihleri arasında Sakarya Havzası' nın Ankara ili sınırları içerisindeki kollarından seçilen istasyonlardan yakalanan Leuciscus cephalus türünün büyüme özellikleri incelenmiştir. Bununla birlikte, farklı ekolojik özellikler gösteren istasyonların fiziksel ve kimyasal paramatreleri belirlenerek, bunların istasyonlardan yakalanan balıkların tür kompozisyonu ve populasyon yoğunlukları üzerindeki etkileri tartışılmıştır. Yapılan su analizleri sonucunda, çalışma alanının su kalitesinin giderek bozulduğu belirlenmiştir ve bu durumun, balıkların tür kompozisyonundan çok populasyon yoğunluktan üzerinde etkili olduğu bulunmuştur. Çalışma sonunda, çevresel faktörlerin değişimine dayanıklı olan Orthrias angorae'mn % 29,70, ekonomik öneme sahip olan Capoeta tinccf nm ise % 28,80'lik bir oranla ihtiyofaunadaki en baskın türler olduğu saptanmıştır. Kirmir Çayı, Ova Çayı ve Aladağ Çayf ndan yakalanan Leuciscus cephalus örneklerinin yaş-boy, yaş-ağırlık ve boy- ağırlık ilişkisi açısından aralannda önemli bir farklılık görülmemiştir. Buna karşın, Aladağ Çayfndaki L cephalus bireylerinin yaşlara göre saptanan ortalama kondisyon faktörü değerlerinin diğer istasyonlardan elde edilen değerlerden düşük olduğu ve bu farkın da istatistiksel açıdan önemli olduğu belirlenmiştir. ABSTRACT In this research, the growth characteristics of Leuciscus cephalus which were caught in the sampling stations that were selected from the Sakarya Basins' tributaries in Ankara, between July 1994 - June 1995, were determined. In relation to this, after identifying the physical and chemical parameters of the sampling stations which show different ecological characteristics, the effect of these parameters on the ichthyofauna and population densities of the fish caught in the sampling station were discussed. As a result of water analysis, it was determined that the water quality of the studied area were becoming polluted and it was found out that this situation affects the population density more than the ichthyofauna. After the research, it was realized that Orthrias angorae and Capoeta tinea with an economic value, which endured the changes of the environmental factors were the dominant species in the ichthyofauna with the rates of (). angorae 29.7% and ( / Unca 28.8%. There was no significant difference revealed between the relations of age- length, age-weight and length-weight of L cephalus samples which were caught in Kirmir Çayı, Ova Çayı and Aladağ Çayı. On the other hand, the average condition factors which were determined according to the ages of the L. cephalus species in Aladağ Çayı were found to be less than the results which were taken from the other sampling stations and it was determined that this difference was important from the statistical aspects. 114

367

oz Bu araştırmada, înci Kefali (Chalcalburnus tarichi) 'nden balık unu üretimi ve üretilen ürünün kalitesi üzerinde çalışılmışdır. Balıklar bütün olarak pişirilmiş, kıyılmış, preslenmiş, kurutularak blenderde parçalanmış ve değirmende öğütülmüşdür. Elde edilen balık ununda fiziksel ve kimyasal analizler yapılmıştır. İnci Kefalinden yapılan balık ununda renk; Ekim ve Kasım örneklerinde koyu san kahve; Mayıs ve Aralık örneklerinde açık sanmsı-kahve olarak belirlenmiştir. Ekim ve Aralık örneklerinde topaklaşmalar olurken, Kasım ve Mayıs örneklerinde topaklaşmaya rastlanmamıştır. Ekim ve Kasım 'da kemik-kılçık oranının Mayıs ve Aralık 'ta üretilenden daha fazla olduğu; Ekim, Kasım ve Aralık ayı örneklerinin Mayıs ayı örneklerinden daha ağır kokulu olduğu belirlenmiştir. Mayıs ayı balık ununun normal balık unu görünümünde, partikül büyüklüğü bakımından homojen olduğu belirlenmiştir. İnci kefalinden üretilen balık unlarında yapılan kimyasal analizlerde, ham protein % 51.65- 56.58; ham kül % 11.22-16.54; ham yağ % 20.44-26.12; nem oram ise %2.68-5.46 arasında belirlenmiştir. Kalsiyum oranı % 6.47-7.79; fosfor oram % 2.56-3.0 arasında bulunmuştur. Elde edilen unda pepsin-HCl de sindirilebilir protein % 51.48, protein sindirilebilirliği ise % 91 olarak belirlenmiştir. Un üretim randımanı % 20 olarak belirlenmiş, undaki fazla yağ ile düşük nem oranının üretim metodundan kaynaklandığı sonucuna varılmıştır. İnci kefalinden balık unu üretiminin ekonomik olacağı, hayvan beslemede emniyetle kullanılabileceği söylenebilir. ABSTRACT In this research, fish meal production from the İnci Kefali (Chalcalburnus tarichi), a local fish species presents in Lake Van, and meal quality were studied. The fish was cooked, pinched, pressed, dried, cracked and grinned by using grinder.Physical and chemical analyses were made on the produced meal. The colours of meals were determined as dark yellow-brown in October and November samples, pale yellowish-brownin May and December samples. While lump formation were observed in October and December production, in May and November there were no lump formation. More fish bone remains was found in October and November than others months. October, November and December products smelled densely while the meal produced in May has charasteristic fish smell. The meal which product in May was to be normal fish meal appearance and has uniform particle composition. At the chemical analyses of fish meal which produced from Chalcalburnus tarichi following values were determined. Crude protein 51.65 - 56.58 %; crude ash 1 1.22 - 16.54 %; crude fat 20.44-26.12 %; moisture 2.68-5.46 % and dry matter 94.54- 97.32 %. In the meals calcium were found as 6.47-7.79 % and phosphorus as 2.56-3.00 %. The meal has 51.48 % pepsin-HCl digestible and digestibility of protein was calculated as 91 %. The production profit of meal was determined as 20 %. It can be concluded that excess crude fat and dry matter originated from production method. It can be also say that meal production from Chalcalburnus tarichi is profitable and the product can be used in animal nutrition confidently. 48

ÖZ Bu çalışmada inci kefalinde (Chalcalburmıs tarichi, Pallas 1811) üreme dönemi öncesi, üreme zamanı ve üreme dönemi sonrası serum inorganik iyon konsantrasyonları, total protein, üre, amonyak (plazmada), bazı enzimler (SGOT, SGPT, LDH) ve hematokrit değerleri incelenmiştir. İnci kefali tatlısuya girdiğinde serum inorganik iyon konsantrasyonlarında, total protein, hepatosomatik indeks, üre ve amonyak (plazmada) seviyelerinde düşme gözlenirken hematokrit, LDH, SGOT ve SGPT değerlerinde artış gözlenmiştir. İnci kefalleri göle döndüklerinde ise serum inorganik iyon konsantrasyonları, total protein, üre ve amonyak seviyeleri yükselme gösterirken, hematokritin düştüğü gözlenmiştir. ABSTRACT In this study, serum inorganic ion concantrations, total protein, LDH, SGOT, SGPT, ürea, ammonia (in plasma) and hematocrit of the Chalcalburrms tarichi (Pallas, 1811) were determined prespawning, during spawning and postspawning. Decreasing in serum ion concantrations, total proetin, hepatosomatic indeks, ürea, ammonia (in plasma) and increasing in LDH, SGOT, SGPT and hematocrit were observed during spawning in Chalcalburrms tarichi migrating to freswater rivers for spawning. When Chalcalburrms tarichi returned to Lake Van increasing in serum inorganic ion concantrations, total protein, urea and ammonia ( in plasma) concantrations and decreasing in hematocrit were observed after spawning. 52