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We evaluated the metabolism of several herbicides and progesterone by two P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica). Expression vectors harboring CYP1A9 and CYP1C1 sequences were introduced into Escherichia coli. E. coli membrane fractions were incubated with each substrate, and the metabolites were analyzed. CYP1A9 and CYP1C1 deethylated 7-ethoxycoumarin and phenacetin, and demethylated chlorotoluron, diuron, and linuron. CYP1C1 specifically hydroxlyated progesterone at the 6β and 16α positions. Five amino acids of CYP1A9 related to substrate binding were selected for mutation analyses [CYP1A9(F128A), CYP1A9(F229A), CYP1A9(F263A), CYP1A9(V387A), and CYP1A9(I391A)]. Two variants, CYP1A9(F229A) and CYP1A9(F128A), changed the ratio of 16α hydroxyprogesterone to 6β hydroxyprogesterone. Among all the variants, CYP1A9(F263A) showed the highest activity towards substrates used. CYP1A9(V387A) and CYP1A9(I391A) showed higher activities than that of CYP1A9 toward progesterone. The substrate specificity of CYP1A9 may be altered by replacing an amino acid related to substrate binding.

In this study, the localization of fluorescent protein (FP) was characterized in the muscles of four species and two subspecies of eels Anguilla anguilla, A. australis, A. bicolor bicolor (b.), A. bicolor pacifica (p.) and A. mossambica in addition to the previously reported A. japonica. The open reading frame of each eel FP was 417 bp encoding 139 amino acid residues. The deduced amino acid sequences among the four species and two subspecies exhibited 91.4–100% identity, and belonged to the fatty-acid-binding protein (FABP) family. The gene structure of eel FPs in A. japonica, A. anguilla, A. australis, A. bicolor b., A. bicolor p. and A. mossambica have four exons and three introns, and were common to that of FABP family. The apo eel FPs expressed by Escherichia coli with recombinant eel FP genes were analysed for the fluorescent properties in the presence of bilirubin. The excitation and emission spectra of holo eel FPs had the maximum wavelengths of 490–496 and 527–530 nm, respectively. The holo eel FPs indicated that the fluorescent intensities were stronger in A. japonica and A. bicolor than in A. mossambica, A. australis and A. anguilla. The comparison of amino acid sequences revealed two common substitutions in A. mossambica, A. australis and A. anguilla with weak fluorescent intensity.

Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227bp, 1194bp and 1155bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9kDa, 44.4kDa and 43.6kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish.

Cytochrome P450 1 (CYP1) is widely used as an indicator of exposure to environmental contaminants. In the study, two full-length complementary DNAs encode for CYP1B1 and CYP1C1 were cloned from medaka liver exposed to 500 ppb β-naphthoflavone for 24 h. CYP1B1 , having 1984 bp, contains an open reading frame of 1551 bp encoding a protein of 517 amino acids; while, CYP1C1 having 2601 bp consists of an open reading frame of 1578 bp encoded for 525 amino acid residues. The highest levels of these CYP1 genes transcript were observed in intestine and the lowest in liver from the fish fed on fuel oil-contaminated feed. Javanese medaka CYP1B1 and CYP1C1 transcripts were detected in the gill, muscle and liver when transferred from seawater to freshwater with the highest level of expression in gill and muscle. Starvations for one week tended to down- regulate the Javanese medaka CYP1 expression. Keywords: Cytochrome P450, Javanese medaka, salinity, starvation, heavy fuel oil, cloning, expression African Journal of Biotechnology , Vol 13(20), 2028-2040

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new cDNA of the CYP1C subfamily encoding CYP1C1 was isolated from Japanese eel (Anguilla japonica) liver after intraperitoneal injection with βnaphthoflavone (BNF). The full-length cDNA obtained (3508 bp) contained a 5' noncoding region of 355 bp, an open reading frame of 1581 bp coding for 526 amino acids, a stop codon, and a 3' noncoding region of 1572 bp. The predicted molecular weight of the protein was approximately 59.33 kDa. The deduced amino acid sequence of Japanese eel CYP1C1 had the lower similarity of 70% with that of killifish CYP1C1 while the higher similarity (79 and 81%) was observed with that of rainbow trout CYP1C2 and -1C1 sequences respectively. It exhibited similarities of 71% with that of Indian medaka CYP1C1 and zebrafish CYP1C2. Also the similarity of 74% was registered with the sequence of three-spined stickleback fish CYP1C1, 1C2 and carp CYP1C2. It showed similarity of 77% with that of Nile tilapia CYP1C1, scup CYP1C1 and scup CYP1C2. The phylogenetic tree showed the newly identified Japanese eel CYP1C1 sequence to be clustered with rainbow trout CYP1C1 and -1C2. Japanese eel CYP1C1 was aligned with the CYP1 sequences and has been deposited in the Gen Bank / EMBL data bank with the accession number AY444748. Quantitative real-time polymerase chain reaction (QRT-PCR) analysis of liver, kidney, intestine and gills revealed a distinct induced expression in all organs studied (283.33, 579.35, 20.96 and 3642.32 respectively).

The CYP1 family, one of the gene families of the CYP superfamily, has four subfamilies deposited in the GenBank/EMBL so far; CYP1A, CYP1B, CYP1C, and the newly identified CYP1D. The metabolic activation and elimination of polyaromatic hydrocarbons, polychlorinated biphenyls, and aryl amines from fish body is largely mediated by the CYP enzymes. A new cDNA of the CYP1B subfamily encoding CYP1B1 was isolated from Japanese eel liver after a single intraperitoneal injection of β-naphthoflavone (BNF). The full-length cDNA obtained was 2985 bp and contained a 5' noncoding region of 294 bp, an open reading frame of 1626 bp coding for 541 amino acids and a stop codon and a 3' noncoding region of 1065 bp. The predicted molecular weight of the protein was approximately 61.27 kDa. The deduced amino acid sequence of Japanese eel CYP1B1 showed 62% similarity to three-spined stickleback CYP1B1 and zebrafish CYP1B1. It exhibited similarities of 66% with that of killifish, Indian medaka and our previously reported carp CYP1B1 and -1B2 while the higher similarities (67 and 69%) of the deduced amino acids was observed with that of Nile tilapia CYP1B1 and rainbow trout respectively. The percent identities of Japanese eel CYP1B1 cDNA showed similarities with those of the reported CYP1Bs of mammals of 57, 57, and 56% for human, rat, and mouse CYP1B1, respectively. Japanese eel CYP1B1 was aligned with the CYP1 sequences and has been deposited in the GenBank/EMBL data bank with the accession number AY518340. The phylogenetic tree constructed using the previously reported CYP1B sequences of mammals and fish suggested the closer relationship of the newly identified Japanese eel CYP1B1 to rainbow trout CYP1B1. QRT-PCR analysis of liver, kidney, gills and intestine revealed a distinct induced expression in liver, kidney and gills (71.93, 3.87 and 539.56 respectively) while the constitutive expression (0.062) was observed in intestine.

The present study aimed to examine the presence of p53 gene mutations in white albino rats fed on repeated fried palm oil. Eighteen sexually mature male Albino rats were used throughout the experiment. Rats were divided randomly into three groups, 6 animals each, namely; control group (CO) which orally administered by distilled water (NO) group which fed on basal diet containing fresh pure palm oil (FO) group which fed on the basal diet containing repeated fried palm oil with a dose of 150 mL kg-1 diet. At the end of the experiment, liver samples were taken, kept at -80°C for genetic alteration studies. Results shows the presence of fifty eight base-pair substitutions mutations which include a total of twelve base pair substitutions arose at A:T base pairs, fourteen base pair substitutions arose at G:C base pairs, Eighteen base pair substitutions at T: A base pairs, fourteen base pair substitutions arose at C:G base pairs. Of the fifty eight substitutional mutations recorded, there were thirty one silent (same sense) mutations and twenty seven missense mutations which causing the substitution of amino acids.

Cytochrome P4501 (CYP1) family enzymes are most active in hydroxylating a variety of environmental contaminants including Polyaromatic Hydrocarbons (PAH), planar polychlorinated biphenyls and arylamines. CYP1B which belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to arylhydrocarbon receptors (AhR) ligands. In this study, a new complementary DNA (cDNA) of the CYP1B subfamily encoding 1B1 was isolated from Nile tilapia ( Oreochromis niloticus ) liver after intracoelomic injection with benzo (a) pyrene (BaP). The full-length cDNA was 2107 base pair (bp) long and contained a 5' noncoding region of 29 bp, an open reading frame of 1527 bp coding for 508 amino acids and a stop codon, and a 3' noncoding region of 551 bp, respectively. The deduced amino acid sequence of Nile tilapia CYP1B1 shows similarities of 79.7, 70.3, 65.7, 65.4, 65.0, and 63.7% with Plaice CYP1B1, Japanese eel CYP1B1, zebra fish CYP1B1, common carp CYP1B1, common carp CYP1B2 and Channel catfish CYP1B1, respectively. The phylogenetic tree based on the amino acid sequences clearly shows tilapia CYP1B1 and Plaice CYP1B1 to be more closely related to each other than to the other CYP1B subfamilies. Furthermore, real-time PCR was used for measuring BaP induction of CYP1B1 mRNA in different organs of tilapia ( O. niloticus ), using β-actin gene as internal control, and the results revealed that there was a large increase in CYP1B1 mRNA in liver (22.8), intestine (2.0) and muscles (1.3). Keywords: Oreochromis niloticus , benzo (a) pyrene, CYP1B1 cDNA, sequence analysis, real-time PCR.

This study planned to isolation and characterization of AKR1A1 cDNA from Bap injected nile tilapia (Oreochromis niloticus), comparison of its characteristic structures with those of other species, characterization of AKR1A1 gene and promoter, and investigation of AKR1A1 mRNA expression in various organs of Bap injected tilapia. The cDNA was 1172 bp long which includes an open reading frame of 975 bp encoding a 324 amino acids protein and a stop codon. The sequence showed 3' and 5' non-coding regions of 179 and 18 bp. The amino acid sequence of O. niloticus AKR1A1 shows similarities of 60, 60, 60.6, 61.2 62.2, and 57.8% with mouse AKR1A1, Norway rat AKR1A1, zebrafish AKR1A1, African clawed frog AKR1A1, human, and yellow perch AKR1A1, respectively. Nucleotide sequence investigation of AKR1A1 gene and 5'-flanking region showed that the structural gene and the 5'-flanking region were approximately 2975 bp and 4006 bp in length, respectively. The protein-coding region contained eight exons, and one additional upstream exon. Real-time polymerase chain reaction (PCR) results showed that the highest level of AKR1A1 expression was found in bile (108.7), followed by kidney (77.9), muscles (37.3), and liver (24.7). mRNA levels of AKR1A1 were almost negligible in gills (0.6) while no detectable (ND) constitutive expression was detected in gut. In conclusion, our results concluded that tilapia AKR1A1 is inducible by BaP and have a significant function in the metabolism of xenobiotics and, therefore, may used as biomarker in fish.

Cytochrome P450 (CYP) enzymes are members of the hemoprotein superfamily, and are involved in the mono-oxygenation reactions of a wide range of endogenous and exogenous compounds in mammals and plants. Characterization of CYP genes in fish has been carried out intensively over the last 20 years. In Japanese pufferfish (Takifugu rubripes), 54 genes encoding P450s have been identified. Across all species of fish, 137 genes encoding P450s have been identified. These genes are classified into 18 CYP families: namely, CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP17, CYP19, CYP20, CYP21, CYP24, CYP26, CYP27, CYP39, CYP46 and CYP51.We pinpointed eight CYP families: namely, CYP1, CYP2, CYP3, CYP4, CYP11, CYP17, CYP19 and CYP26 in this review because these CYP families are studied in detail. Studies of fish P450s have provided insights into the regulation of P450 genes by environmental stresses including water pollution. In this review, we present an overview of the CYP families in fish.

CYP1C is the newest member of the CYP1 family of P450s; however, its physiological significance, inducers, and metabolic functions are unknown. In this study, a new complementary DNA of the CYP1C subfamily encoding CYP1C1 was isolated from Nile tilapia ( Oreochromis niloticus ) liver after intracoelomic injection with benzo-a-pyrene (BaP). The full-length cDNA was 2223 base pair (bp) long and contained an open reading frame of 1581 bp encoding a protein of 526 amino acids and a stop codon. The sequence exhibited 3′ non-coding region of 642 bp. The deduced amino acid sequence of O. niloticus CYP1C1 shows similarities of 86, 82.5, 79.7, 78.7, 77.8, 75.5, 69.6 and 61.3% with scup CYP1C1, killifish CYP1C1,1C2, Japanese eel CYP1C1, zebra fish CYP1C1, common carp CYP1C1, scup CYP1C2, common carp CYP1C2 and zebra fish CYP1C2, respectively. Phylogenetic tree based on the amino acids sequences clearly shows tilapia CYP1C1 and scup CYP1C1 to be more closely related to each other than to CYP1C genes from other species. Furthermore, for measuring BaP induction of CYP1C1 mRNA in different organs of tilapia ( O. niloticus ), β-actin gene as internal control was selected based on previous studies to assess their expression variability. Real time RCR results revealed that there was a large increase in CYP1C1 mRNA in liver (43.1), intestine (5.1) and muscle (2.4).

Through the use of a number of bioconversion experiments we demonstrated that P450 proteins (CYP1A9 and CYP1C1) from Japanese eel ( Anguilla japonica ) metabolized a number of herbicides and the drug phenacetin. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolites were extracted and analyzed by high-performance liquid chromatography. Proteins CYP1A9 and CYP1C1 metabolized 50 nmol of the drug phenacetin to yield 12.1 and 1.1 nmol of product (acetaminophen), respectively. Further incubation of CYP1A9 with 50 nmol of the herbicides chlorotoluron, diuron, linuron, simazine, or atrazine yielded 16.5, 18.5, 7.3, 1.6, or 0.8 nmol of product, respectively. CYP1C1 also metabolized linuron, diuron, and simazine yield 5.4, 4.6, or 0.7 nmol of product, respectively. Next, polyclonal antibody was isolated by immunizing with two conjugated-peptides (amino acid residues 272–290 and 294–310) of CYP1A9. This antibody did not recognize human CYP1A2 or CYP1C1. Western blotting using the antibody revealed one band in the livers of Japanese eel and tilapia ( Oreochromis niloticus ). Theses results suggest that CYP1A9 and CYP1C1 metabolize herbicides, and that CYP1A9 is an useful biomarker of contamination when detected with this antibody.

Apolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.

Green fluorescent protein (GFP) from eel (Anguilla japonica) muscle (eelGFP) is unique in the vertebrates and requires bilirubin as a ligand to emit fluorescence. This study was performed to clarify the physiological function of the unique GFP. Investigation of susceptibility to oxidative stress was carried out using three types of cell lines including jellyfish (Aequorea coerulescens) GFP (jfGFP)-, or eel GFP (eelGFP)-expressing HEK293 cells, and control vector-transfected HEK293 cells. Binding of eelGFP to bilirubin was confirmed by the observation of green fluorescence in HEK293-eelGFP cells. The growth rate was compared with the three types of cells in the presence or absence of phenol red which possessed antioxidant activity. The growth rates of HEK293-CV and HEK293-jfGFP under phenol red-free conditions were reduced to 52 and 31% of those under phenol red. Under the phenol red-free condition, HEK293-eelGFP had a growth rate of approximately 70% of the phenol red-containing condition. The eelGFP-expressing cells were approximately 2-fold resistant to oxidative stress such as H2O2 exposure. The fluorescence intensity partially decreased or disappeared after exposure to H2O2, and heterogeneous intensity of fluorescence was also observed in isolated eel skeletal muscle cells. These results suggested eelGFP, but not jfGFP, coupled with bilirubin provided the antioxidant activity to the cells as compared to non-bound free bilirubin.

Cytochrome P450 family 1 (CYP1A) subfamily genes are the most well studied CYP genes in vertebrates; however, information on CYP1A genes in Javanese medaka is relatively scarce. In the present study, full-length cDNA of CYP1A was cloned from the fish liver exposed to 500 ppb β-naphthoflavone for 24 h, which is 2439 bp contained in an open reading frame of 1593 bp encoding a protein of 530 amino acids. Real time polymerase chain reaction (RT-PCR) was used to measure the quantitatively tissue expression of the gene by environmental stress conditions. The results indicate that the highest levels of the CYP1A gene transcript was in intestine and the lowest in liver of the fish that fed on fuel oil-contaminated feed. Javanese medaka CYP1A transcripts were detected in the gill, muscle and intestine when transferred from seawater to freshwater with the highest level of expression in gill and muscle. CYP1A gene expression in the tissues tends to be down-regulated in Javanese medaka starved for one week. Keywords: Cytochrome P450, CYP1A, Javanese medaka, heavy fuel oil, salinity, starvation, cloning, expression African Journal of Biotechnology Vol 13(18), 1898-1909

Edwardsiella tarda is one of the major pathogenic bacteria affecting both marine and freshwater fish species. Sialidase NanA expressed endogenously in E. tarda is glycosidase removing sialic acids from glycoconjugates. Recently, the relationship of NanA sialidase activity to E. tarda infection has been reported, however, the mechanism with which sialidase NanA aids the pathogenicity of E. tarda remained unclear. Here, we comprehensively determined the biochemical properties of NanA towards various substrates in vitro to provide novel insights on the potential NanA target molecule at the host cell. GAKS cell pretreated with recombinant NanA showed increased susceptibility to E. tarda infection. Moreover, sialidase inhibitor treated E. tarda showed a significantly reduced ability to infect GAKS cells. These results indicate that NanA-induced desialylation of cell surface glycoconjugates is essential for the initial step of E. tarda infection. Among the natural substrates, NanA exhibited the highest activity towards 3-sialyllactose, α2-3 linked sialic acid carrying sialoglycoconjugates. Supporting this finding, intact GAKS cell membrane exposed to recombinant NanA showed changes of glycoconjugates only in α2-3 sialo-linked glycoproteins, but not in glycolipids and α2-6 sialo-linked glycoproteins. Lectin staining of cell surface glycoprotein provided further evidence that α2-3 sialo-linkage of the N-linked glycoproteins was the most plausible target of NanA sialidase. To confirm the significance of α2-3 sialo-linkage desialylation for E. tarda infection, HeLa cells which possessed lower amount of α2-3 sialo-linkage glycoprotein were used for infection experiment along with GAKS cells. As a result, infection of HeLa cells by E. tarda was significantly reduced when compared to GAKS cells. Furthermore, E. tarda infection was significantly inhibited by mannose pretreatment suggesting that the bacterium potentially recognizes and binds to mannose or mannose containing chains following desialylation. Together, these results suggest that E. tarda may employ endogenous NanA to desialylate α2-3 glycoproteins on host cells, thus revealing one of the potential binding molecules during infection.

The cytochrome P450 (CYP) represents a large group of microsomal monooxygenases that catalyze drugs as well as a host of lethal environmental contaminants such as dioxins, leading to either detoxification and excretion from the animal or generation of carcinogenic intermediates. In the present study two forms of cDNA were cloned (Eu MC1 and Eu MC2) for European eel CYP1A genes by polymerase chain reaction (PCR) techniques. The cDNA of Eu MC1 was 3368 bp long coding 521 amino acid residues, and that of Eu MC2 was 2464 bp long coding 517 amino acid residues. Identities of deduced amino acid sequences between Eu MC1 and Japanese eel CYP1A1 and that between Eu MC2 and the second form of Japanese eel CYP1A were 98% and 97%, respectively, showing decisively that Eu MC1 and Eu MC2 are orthologous to Japanese eel CYP1A1 and the second form of CYP1A, respectively. A striking difference between the two eel species was that the Eu MC1 peptide was two amino acid residues longer than that of the Japanese eel CYP1A1. Existence of two loci of CYP1A in Japanese and European eels may suggest that the two forms of CYP1A exist widely among the eel species, because the divergence between the two eel species has been shown to be close to the basal divergence among eels. The identities in CYP1A may help to estimate genetic distance between European and Japanese eels.

The putative CYP1A1 gene was isolated from a genomic library of eel (Anguilla japonica) chromosomal DNA. Sequence analysis of 8184 bp showed that the clone obtained was the structural gene of CYP1A1, which consisted of seven exons and six introns in a region approximately 5800 bp in length, as well as a 5' upstream region of about 2300 bp. The initiation codon was found not in the first exon, but in the second exon, as was reported for the CYP1A genes of mammals and fish. The GC box, CAAT box, and TATA box were located proximately to the first exon, at 110, 62, and 38 bp upstream of the transcriptional start site, respectively. We found six core sequences of xenobiotic-responsive elements (XREs) in the 5' upstream region, and also one in the first intron. All the XRE core sequences in the 5' upstream region were unique in their localization in the sense strand. The XRE core sequence in the first intron was in the antisense strand.

Transcription of the CYP1A1 genes in mammals and fish is stimulated by polyaromatic hydrocarbons. DNA sequencing analysis revealed that CYP1A1 gene in eel (Anguilla japonica) contains two kinds of putative cis-acting regulatory elements, XRE (xenobiotic-responsive element) and ERE (estrogen-responsive element). XRE is known as the enhancer that is responsible for the inducibility of the genes of CYP1A1 and some other drug-metabolizing enzymes. In the eel CYP1A1 gene, XRE motifs are distributed as follows: five times in the region from -2136 to -1125 bp, XRE(-6) to (-2); once in the proximal basal promoter region, XRE(-1); and once in the first intron, XRE(+1). The region between XRE(-2) and XRE(-1) contains three ERE motifs. To investigate the function of the cis-acting regulatory elements in the eel CYP1A1 gene, recombinant plasmids prepared with its 5' upstream sequence and the structural gene for luciferase were microinjected into fertilized eggs of medaka at the one-cell stage. Hatched fry were treated with 3-methylcholanthrene, and the transcription efficiency was assayed using competitive polymerase chain reaction analysis. Deletion of the region containing the five XREs, XRE(-6) to XRE(-2), and the point mutation of XRE(-1) reduced the inducible expressions by 75% and 56%, respectively, showing apparent dependency of the drug induction on the XREs. Constitutive expression, however, was not significantly affected by deletion or disruption of the XREs. When the region between XRE(-2) and XRE(-1) containing no XREs but three ERE motifs was internally deleted, the inducible expression and the constitutive expression were reduced by 88% and 75%, respectively. Replacement of this region with a partial fragment of eel CYP1A1 complementary DNA, with slight alteration of the distance between the five XREs and XRE(-1), reduced the inducible expression and the constitutive expression by 91% and 60%, respectively. These results strongly suggest that not only XRE but also other regulatory elements, possibly ERE, play an important role in induced and constitutive expressions of the eel CYP1A1 gene.

We characterized the genes of the male-specific mouse steroid 16α-hydroxylase (C-P-45016α) and the female-specific mouse steroid 15α-hydroxylase (P-45015α) within two distinct gene families. In spite of the high structural identities within each family, the expression of the hydroxylase genes is uniquely regulated. Moreover, the other family members encode the P-450s which are structurally very similar to the hydroxylases but are not able to catalyze steroid hydroxylase activities. For example, only a single amino acid substitution creates steroid 15α-hydroxylase activity in another family-member P-450coh, which catalyzes coumarin 7-hydroxylase but little steroid hydroxylase activity. It appears, therefore, that the mouse P-450 gene families evolved through gene duplication and selective mutation to create new P-450s structurally as well as to establish novel regulatory elements for the gene expressions.Key words: cytochrome P-450, steroid hydroxylase, site-directed mutagenesis, evolution, gene regulation.

Cytochrome P450 (CYP) genes, which make up a large gene superfamily, are known to play an important role in drug metabolism. The CYP1 family, one of the gene families of the CYP superfamily, has three subfamilies of genes whose sequences have been deposited in the GenBank/EMBL thus far: CYP1A, CYP1B, and CYP1C. Mammals as well as fish confront numerous foreign chemicals in the environment that may accumulate to toxic levels unless they are metabolized and eliminated by processes largely mediated by CYP enzymes. A new complementary DNA of the CYP1C subfamily encoding CYP1C2 was isolated from the carp liver after a single intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 198 bp, an open reading frame of 1575 bp coding for 524 amino acids and a stop codon, and a 3' noncoding region of 531 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The amino acid sequence deduced from the carp CYP1C2 sequence showed a similarity of 76.6% with that deduced from our previously reported carp CYP1C1. It exhibited similarities of 77.3, 73.7, and 76.4% with those deduced from scup CYP1C2, scup CYP1C1, and Japanese eel CYP1C1 sequences, respectively. Carp CYP1C2 cDNA showed similarities with reported CYP1Bs of teleosts and mammals, namely, 47.6, 45.3, 45.7, 44.0, and 44.6% for carp, plaice, human, rat, and mouse CYP1B1s, respectively, while it exhibited a similarity of 49.0% with carp CYP1B2. The carp CYP1C2 sequence was aligned with the CYP1 sequences and has been deposited in the GenBank/EMBL data bank with the accession number AY437777. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of CYP1C with CYP1B than with CYP1A. The tree showed possibile existence of CYP1C subfamily genes in mammalian species. Northern blot analysis of the liver, intestines, kidneys, and gills revealed a distinct induced expression only in the kidneys, with no detectable constitutive expression in the other organs studied.

We indicated that two P450s (1A9 and 1C1) from Japanese eel (Anguilla japonica) metabolized 7-ethoxycoumarin, 7-ethoxyresorufin, and flavanone. At first, we constructed expression vectors for two types of P450 (1A9 and 1C1). The reduced CO-difference spectra of Escherichia coli cells transformed with these plasmids showed Soret peaks (450 nm) that were typical of P450s. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolite(s) were extracted and analyzed by high-performance liquid chromatography and spectrofluorometer. Incubation of 50 nmol 7-ethoxyresorufin with P450 1C1 yielded 0.773 nmol of deethylated product, whereas 50 nmol 7-ethoxycoumarin resulted in 4.76 nmol. P450 1A9 metabolized 50 nmol of 7-ethoxyresorufin and 7-ethoxycoumarin to yield 6.54 and 20.9 nmol of deethylated product, respectively. Incubation of 50 nmol flavanone with P450 1C1 yielded 1.46 nmol and 0.69 nmol of products, whereas 50 nmol flavanone with P450 1A9 resulted in 1.10 nmol. In this system, 4'-hydroxy flavanones were formed by P450 1A9 and P450 1C1. P450 1A9 also metabolized 50 nmol of 17 beta-estradiol to yield 4.25 nmol of product. In this system, 2-hydroxy estradiol was formed by P450 1A9 using 17 beta-estradiol as a substrate. This study is the first to identify the substrates that P450 1C1 and 1A9 metabolize.

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new complementary DNA of the CYP1C subfamily encoding CYP1C1 was isolated from carp liver after intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 244 bp, an open reading frame of 1572 bp coding for 524 amino acids, a stop codon, and a 3' noncoding region of 965 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The deduced amino acid sequence of this cDNA was 82.1% and 80.2% similar to Japanese eel and scup CYP1C1 sequences, respectively, while it exhibited a similarity of 74.9% with the scup CYP1C2 sequence. The deduced amino acid sequence of carp CYP1C1 showed similarities with those of the reported CYP1B1s of teleosts and mammals of 48.4, 48.8, 48.2, 48.6, 45.3, and 45.5% for carp CYP1B1, carp CYP1B2, plaice CYP1B1, and human, rat, and mouse CYP1B1, respectively. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of the CYP1C subfamily to CYP1B than to CYP1A. The tree showed the possibility of the existence of CYP1C subfamily genes in mammalian species. Northern blot analysis for the liver, intestine, gills, and kidney showed no detectable induced expression but constitutive expression in the gill organs.

Some forms of cytochrome P450 (CYP) genes are known to be induced by xenobiotics such as dioxins. Induction of the CYP1A gene is mediated by an aryl hydrocarbon receptor (AHR) which binds to a specific nucleotide sequence called a dioxin-responsive element (DRE) located in the 5' enhancer region of the gene. Functional analysis of the regulatory region of the eel CYP1A gene had shown that a 654-bp region near the basal promoter, containing no DREs but three motifs that resemble estrogen-responsive element (ERE) halfsites, contributes substantially to the induced expression. Considering the importance of non-DRE elements in CYP1A gene induction, we investigated the role of ERE-like motifs using a point mutation technique. The regulatory region of the eel CYP1A gene was identified by creating mutations in all three ERE half-sites simultaneously or individually. The regulatory region constructs were cloned upstream of the luciferase gene in an expression vector which was microinjected into medaka ova. Expression of luciferase as a foreign gene in medaka fry exposed to 3-methylcholanthrene (3-MC)-treated feed was measured by competitive PCR analysis. Mutation in all the three ERE half-sites reduced expression to 10%. Mutation in ERE(-2) or ERE(-3) reduced the expression to 50%, whereas mutation in ERE(-1) did not affect the induction. The two functional half-sites of the eel CYP1A gene are palindromic to each other and appear to constitute the full-ERE.

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new complementary DNA of the CYP1B subfamily encoding CYP1B2 was isolated from carp liver after intraperitoneal injection with 3-methylcholanthrene (3-MC). The obtained full-length cDNA contained a 5'noncoding region of 161 bp, an open reading frame of 1593 bp coding for 530 amino acids and a stop codon, and a 3'noncoding region of 1457 bp. The predicted molecular weight of the protein was approximately 60.2 kDa. The deduced amino acid sequence of this cDNA was 91% similar to that of our previously reported carp CYP1B1; its similarities with those of the reported CYP1B1s of teleosts and mammals were 60.8, 54.4, 50.8, and 51.4% for plaice, human, rat, and mouse, respectively. The phylogenetic tree of fish and mammalian CYP1 sequences constructed by the protein maximum-likelihood method suggested a relatively recent divergence of CYP1B2 from CYP1B1 in the ancestor of carp and closely related species. Despite the structural similarity of CYP1B2 with CYP1B1, which showed induced expression in 3-MC-treated liver, intestine, and gills with marked constitutive expression in gills, CYP1B2 revealed induced expression in gills but not in liver or intestine, and no detectable constitutive expression in the tissues studied.

Cytochrome P450 (CYP) genes, which make up a large gene superfamily, are known to play an important role in drug metabolism. The levels of expression of CYP genes in the tissue of fish inhabiting polluted areas have been used extensively in biomonitoring studies as indicators of dioxin pollution. Complementary DNA of cytochrome CYP1B1 was isolated from carp (Cyprinus carpio) liver 24 h after the injection of 3-methylcholanthrene (3-MC). The full length cDNA obtained contained a 5' noncoding region of 178 bp, an open reading frame of 1593 bp coding for 530 amino acids and a stop codon, and a 3' noncoding region of 1599 bp. The predicted molecular weight of the encoded protein was approximately 60.2 kDa. The deduced amino acid sequence exhibited an identity of 60.6% with reported CYP1B sequences of plaice CYP1B, and of 52.4, 51.4, and 50.3% with human, rat, and mouse CYP1B1s, respectively. It exhibited similarities of 48.4 and 47.3% with scup CYP1C2 and -1C1 sequences. The percent identities with CYP1A sequences showed lower values in the range from 35.3 to 39.5% with mammals and teleosts. The phylogenetic tree of the CYP1 family members constructed by the protein maximum likelihood method indicates that the carp CYP1B1 and plaice CYP1B share a common ancestry with the mammalian CYP1B1s. Carp treated with 3-MC showed expression of CYP1B1 in liver, intestine and gills with distinct induction except for the gills that showed marked constitutive expression. The presence of two successive signals in treated gills at low stringency hybridization may suggest the existence of another CYP1B member that is expressed in the gills of carp.

Complementary DNA of cytochrome P-450 CYP1A, in addition to CYP1A1, has been isolated from Japanese eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5′ untranslated region of 66 bp, an open reading frame of 1554 bp coding for 517 amino acids and a stop codon, and a 3′ untranslated region of 1166 bp. The predicted molecular weight of the Japanese eel CYP1A was approximately 58.5 kDa. The nucleotide sequence exhibited identities with the reported CYP1A1 sequences of 77% for Japanese eel, 75% for rainbow trout, 72% for scup, plaice, and butterfly fish, and 71% for toadfish. The deduced amino acid sequence exhibited identities with the reported CYP1A1 sequences of 78% for Japanese eel, 77% for rainbow trout, 75% for scup, 74% for toadfish, 73% for plaice, and 72% for butterfly fish. The novel eel CYP1A obtained had less similarity to the other teleost CYP1A1 proteins (72%–78%) than that of the eel CYP1A1 (74%–80%). When compared with mammalian CYP proteins, the novel eel CYP1A was more similar to the CYP1A1 proteins (54%–56%) than to the CYP1A2 proteins (50%–53%). The phylogenetic tree of the teleost CYP1A genes constructed using the maximum likelihood method suggested that the novel eel CYP1A is ubiquitous among the Anguilliformes.

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.

The gene family cytochrome P450 (CYP) 1A is ubiquitous among animals, and CYP1A1 genes have been identified in teleost and mammals. We isolated another CYP1A gene, which is inducibly expressed by exposure to 3-methylcholanthrene, from the genomic library of the eel (Anguilla japonica). The genomic clone obtained, approximately 17,500 bp in length, contained the structural gene of the CYP1A and a 5' upstream region of about 1600 bp. Sequence analysis of the 5' upstream region and the first and second exons revealed that the initiation codon was in the second exon, as in the CYP1A genes reported for mammals and teleosts. CAAT and TATA boxes were found 51 and 29 bp upstream from the transcriptional start site, respectively. Unlike the CYP1A1 genes of eel and other animals, we found no xenobiotic responsive element (XRE) core sequences in the 5' upstream region studied (1631 bp) or in the first intron, whereas three peculiar regions, each composed of multiple repeats of the trinucleotide TAA, were found between 824 and 1356 bp upstream from the transcriptional start site. Absence of XRE core sequences and presence of the multiple repeats in the 5' upstream region sequenced may suggest that the expression mechanism of the eel CYP1A gene is somewhat different from the mechanisms of other reported CYP1A genes.

Cytochrome P450 1A (CYP1A) complementary DNA was isolated from eel (Anguilla japonica) liver treated with 3-methylcholanthrene. The cDNA contained a 5' untranslated region of 163 bp, an open reading flame of 1560 bp coding for 519 amino acids and a stop codon, and a 3' untranslated region of 1730 bp. The predicted molecular weight was approximately 58.4 kDa. The deduced amino acid sequence exhibited identities with reported CYP1A sequences of 80% for rainbow trout, 79% for scup, 76% for plaice and butterfly fish, and 74% for toadfish. When compared with mammalian CYP proteins, the eel CYP1A was more similar to CYP1A1 (54%-56%) than to CYP1A2 (49%-52%). Northern and Southern blot analyses showed two distinct bands, suggesting the existence of another 3-methylcholanthrene-inducible CYP1A gene in eel.

In eel (Anguilla japonica), exposure to polyaromatic hydrocarbons such as 3-methylcholanthrene leads to induction of two CYP1A enzymes, CYP1A1 and CYP1A6. We studied the time course and tissue specificity of induction of messenger RNAs for CYP1A1 and CYP1A6 in eel by administering 3-methylcholanthrene intraperitoneally. In both cases, the drug induced a rapid increase of mRNAs and biphasic expression. In the liver, mRNA levels of CYP1A1 and CYP1A6 increased 22-fold at 3 hours and 27-fold at 6 hours after the administration, respectively, showing initial peaks in the induction. After the initial inductions, mRNA levels decreased unexpectedly. Following these temporary decreases, the mRNA levels again increased and reached levels that were 35 and 41 times the basal levels at 24 hours after administration, respectively. CYP1A1 and CYP1A6 resembled each other also in the tissue specificity of gene expression; the expression levels were liver ≫ gill > intestine > kidney. The rapid induction, the biphasic expression, and the tissue-specific expression were common features of gene expression in CYP1A1 and CYP1A6 and may come from common structures of the regulatory regions of the two genes.

Commercial preparations of clupeine and salmine sulfates were fractionated into their components by column chromatography and were separated into three (YI, YII and Z) and two (S-A and S-B) fractions respectively. Amino acid composition of the purified fractions showed that they were different quantitatively and qualitatively to each other. Clupeine fraction Z and salmine fraction S-B were found to be richer in arginine content than that of other fractions. The study of antibacterial action of the fractions showed that the fractions containing higher amount of arginine had lower MIC values than that of the whole protamine and other fractions. Although the fractions Z and S-B have shown higher antibacterial action, their MIC values were close to that of whole clupeine and salmine. In general the fractions have shown higher antibacterial action in broth media than that of agar media.

Inhibitory effect of clupeine and salmine sulfates on the growth from the spores of Bacillus sub-tilis and Bacillus licheniformis was investigated. These two protamines were found effective in pre-venting the growth from the spores in both agar and broth media. The level of protamine required to inhibit the growth was found to be more or less dependent on the type of media used and the pH. In general MIC of the protamines ranged from 100 to 150μg/ml in nutrient agar and 10 to 50μg/ml in broth but higher protamine level was required in nutritionally rich Heart Infusion Broth and Penassy Broth. Inhibitory effect of protamine was more profound in the neutral and alkaline pH than in the acidic condition. Filter sterilized and autoclave sterilized protamine had the same inhibitory effect. Spore density in the medium influenced the required level of protamine for preventing the growth from the spores.

Ethanol stimulated gluconeogenesis not only from pyruvate, but also from lactate in the isolated liver cells of the eel. Concentrations of lactate and pyruvate were not changed significantly by the addition of ethanol. Acetate stimulated gluconeogenesis from pyruvate in hepatocytes of the eel. In gluconeogenesis from pyrucate, lactate concentration was increased 2-fold and pyruvate concentration was not changed by the addition of acetate. Lactate release from the perfused liver in gluconeogenesis from pyruvate was also investigated. Little lactate release was observed. Effects of ethanol and acetatc on gluconeogenesis in the eel liver was different form that in mammalian liver. The reasons for these differences were discussed in connection with the lactate dehydrogenase in the eel liver.

Two halophilic strains of Bacillus B3 and Bacillus C1 were isolated from Burmese fish sauce and Chinese fish sauce in Sehgal and Gibbons Complex (SGC) medium with 3M NaCl and in nutrient broth (NB) medium with 2M NaCl respectively. Growth nature and protease formation of two bacteria in different NaCl concentration ranging from 0M to 4M were investigated. Bacteria were cultured in coresponding medium (80ml) with different NaCl concentration at 30°C under reciprocal shaking and 5ml of slurry was taken out every dry and turbidity was measured against a blank. The slurry was centrifuged at ×10000g for 15min to separate the microorganisms and dialyzed with 400 fold water for 24h. Protease activity of dialyzed solution was measued by using 1% casein solution. Both bacteria were found out as capable of growth in medium of 4M NaCl. Maximal growth of Bacillus B3 was obtained in a medium containing 1-2M NaCl and that of Bacillus C1 was obtained in 0-3M NaCl. The protease formation of Bacillus B3 was highest in a medium containing 2M NaCl while that of Bacillus C1 was highest in a medium containing 1M NaCl. The enzyme produced from Bacillus B3 had more resistance to NaCl than that produced from Bacillus C1.

Two types of fish protamines (clupeine sulfater and salmine sulfate) were tested for their antibacterial action on a number of bacterial strains. All the Gram-positive organisms studied were more or less sensitive to these protamies, while there was little or no effect on Gram-negatives. The action was almost same in the two pootamines. According to MIC values, Bacillus coagulans and Bacillus megaterium were the most sensitive organims. The protamines were bactericidal against five out of six bacillus organisms studied and the organism licheniformis was capable to resist the bactericidal action of the protamines.

Optimal pH for reductive amination and glutamate oxidation were 8.0 and 8.5, respectively. The enzyme dept in 0.1M phosphate buffer (pH 6.0) at 8°C for 4 days had 80% of the initial activity, whereas that in 0.1M Tris-CI (pH 8.0) had negligible activity. Km values for NAD+ and glutamate were 0.45 and 3.7mM, respectively. Leucine, methionine, phenylalanine, and valine activated the enzyme in the direction of glutamate formation. Malate and succinate inhibited the enzyme. Effects of nucleotides and divalent ions on the enzyme activity were also examined and the role of the enzyme for gluconeogenesis in the eel liver was discussed.

The antifungal properties of salmine sulfate were tested against 15 strains of molds by determining sensitivity, minimum inhibitory concentration and percent growth inhibition. Out of 15 strains 10 organisms were found to be sensitive to salmine sulfate at a concentration of 500μg per ml medium. The salmine sulfate did not support the growth of mucor species at a concentration of 500μg per ml. The genous Rhizopus was also found sensitive to salmine sulfate. The MIC of the tested molds showed that Eurotium repens and Myceliophthora lutea were most sensitive with 250μg salmine per ml medium. These two species were also lowest in percent growth inhibition of 400μg salmine per ml broth medium. All cultures except Aspergillus niger showed reduction of mycelial weight indicating an inhibitory effect of salmine sulfate on the growth of molds. The growth of Aspergillus niger was not inhibited by salmine sulfate.

Accelerated production of fish sauce by adding some halophilic bacteria was studied. Sardines (200g) were cut into 2-3cm pieces, uneviscerated and well mixed with 50g of salt. Bacillus C1 and Bacillus C6, isolated from Chinese fish sauce and strain No. 3-19-1, isolated from sand of Kinko Bay were cultured in Sehgal and Gibbons Complex (SGC) medium with 3M NaCl and pH 6.6-6.8. The bacteria were harvested on the day of their maximal growth. Each of the bacterial slurry (12.5ml) was added to the mixture of fish pieces and salt and put into glass jar. To the control without bacteria, 12.5ml of solution containing NaCl and MgSO4, the concent-rations of which were identical with the cultural medium was added. Progress in proteolysis of fish sauce was investigated by determining pH, total soluble nitrogen content, amino-type nitrogen content and volatile basic nitrogen content of fish sauce throughout the fermentation process. Bacillus C1 showed remarkable effect on accelerating the process. The liquefaction step could be finishedwithin 3 months. Ripening at various conditions was investigated and it was observed that volatile organic acids appeared within one month in the sample ripened at 30°C under reciprocal shaking. Free amino acids of fish samples were determined and it was recognized that there was no great difference in free amino acid profile of control and that of bacteria-added samples.

Glutamate dehydrogenase (EC 1.4.1.2-4) has been purified from the acetone powder of eel liver; the process utilized affinity chromatography with GTP-Sepharose in the final step. The homogeneity of the enzyme was proved by 1) a single protein-staining band in polyacrylamide gels, 2) a single symmetrical Schlieren peak in the ultracentrifuge, and 3) constant specific activity in the chromatogram obtained by GTP-Sepharose column. Sedimentation analysis gave a S020, w value of 12.3. The enzyme had a molecular weight of 315, 000, as determined by polyacrylamide gel electrophoresis. In gel electrophoresis, the enzyme showed little mobility if ADP was not added in sample, spacer, and separate gel. Amino acid analysis showed that the ratio of quantities of arginine, lysine, and tyrosine to the total residues of amino acids in the eel enzyme were rather higher than those in bovine, rat, and tuna enzymes.

Two cDNAs, pc16 alpha-2 and pc16 alpha-25, which encode P-450s from within the mouse, male-specific steroid 16 alpha-hydroxylase (C-P-450(16 alpha)) gene family, were transfected into COS-1 cells in order to study catalytic activities of the expressed P-450s. pc16 alpha-2 was shown previously to encode the growth hormone dependent and androgen-dependent C-P-450(16 alpha) in adult male mice (Wong et al., 1987). The sequence of pc16 alpha-25-encoded P-450 (P-450cb) was identical with gene cb within the C-P-450(16 alpha) family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-450(16 alpha). We expressed both P-450s by transfecting their cDNAs into COS-1 cells and found that steroid 16 alpha-hydroxylase activity was catalyzed by C-P-450(16 alpha) but not by P-450cb. In addition to testosterone, progesterone and estradiol were hydroxylated specifically at the 16 alpha-position by the expressed C-P-450(16 alpha). The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P-450(16 alpha). We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P-450(16 alpha) molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16 alpha-hydroxylase activity.

Porcine heart adenylate kinase can be separated into acidic and basic components by electrofocusing, and the latter is identical with the skeletal muscle adenylate kinase. The acidic adenylate kinase was purified approximately 3600-fold from porcine heart extract in an overall yield of 59% by DEAE-cellulose chromatography, substrate elution from a column of phosphocellulose, affinity chromatography on a column of blue dextran-Sepharose 4B, and gel filtration. The purified acidic enzyme had a specific activity of 1100 units per mg of enzyme, and was found to be a homogeneous protein in all the procedures used, i.e. disc and sodium dodecylsulfate electrophoresis, and gel filtration. However, electrophoretic variants were observed on isoelectrofocusing and this variance may depend on the formation of protein-ampholine complexes. The purified acidic enzyme had a molecular weight of 26 900 and an optimum pH of 5.8 in the forward reaction (ATP + AMP 2ADP). The amino acid composition of the acidic enzyme was found to be 22 aspartic acid or asparagine, 13 threonine, 14 serine, 29 glutamic acid or glutamine, 17 proline, 17 glycine; 22 alanine, 4 half-cystine, 13 valine, 8 methionine, 13 isoleucine, 24 leucine, 4 tyrosine, 7 phenylalanine, 5 histidine, 19 lysine, 14 arginine and no tryptophan, totaling 245 residues. The enzyme had two – SS – bonds but no free – SH, and was not inhibited at all by 5,5′-dithio-bis(2-nitrobenzoic acid). From these results, it may be concluded that the acidic adenylate kinase of porcine heart is similar to liver mitochondrial adenylate kinase.