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Rice bran lectins, named as RBA1 and RBA2, were isolated from Oryza sativa in two chromatography steps: affinity chromatography and cation-exchange chromatography. RBA1 was found to be composed of a covalently linked heterodimer of 20- and 12-kDa subunits, and RBA2 was a noncovalently linked dimer of 12-kDa subunits. Both RBA1 and RBA2 bound to desialylated complex glycoproteins such as fetuin, α1-acid glycoprotein, and transferrin, and agalactosylated complex glycoproteins such as agalacto fetuin, agalacto-α1-acid glycoprotein, and agalacto-transferrin, in addition to chitooligosacchrides. RBAs were heat stable up to 80 °C and stable at pH 4–10. RBA1 increased the transport of the fluorescent marker, rhodamine 123, which is known to be transported via the P-glycoprotein-mediated efflux pathway across human intestinal Caco-2 cell monolayers. Furthermore, RBA1 itself was transported to the basolateral side of the monolayers via an endocytotic pathway.

Dioscorea batatas tuber lectin 1 (DB1) is a storage protein isolated from yam tuber and has been shown to be a mannose-binding lectin. Here we produced transgenic rice plants expressing cDNA of DB1 under the control of phloemspecific promoter of rice sucrose synthase-1 gene. DB1 accumulated at a level of 0.07% of total soluble protein. We then evaluated its efficacy for brown planthopper (BPH). After releasing the first instar of BPH on the transgenic rice plants, the number of survived BPH adults was reduced up to 30% compared to that of wild-type rice. The number of the next generation BPH was suppressed to 22% on average in the seven most-resistant plants compared to that of wild-type rice plants when female adult BPH was inoculated. These results demonstrated that DB1 is effective to confer BPH resistance in terms of decreased survival and fecundity.

Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families.

Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).

Angiotensin-I-converting enzyme (ACE) plays a crucial role in the crisis of hypertension. Some peptides that originate from protease hydrolysates are known to suppress ACE activity in vitro and in vivo. Here, we investigated whether trypsin hydrolysate of oyster Crassostrea gigas showed hypotensive activity and ACE inhibition. The hydrolysate significantly suppressed systolic blood pressure and ACE activity in spontaneously hypertensive rats following a one-shot oral administration and a long-term feeding experiment lasting 9 weeks. Each hydrolysate from oyster tissue showed ACE inhibitory activity, indicating the hypotensive effect was due to synergism. One potent ACE inhibitory peptide, Asp-Leu-Thr-Asp-Tyr, was identified from the hydrolysate of the striate muscle, and the peptide exhibited hypotensive activity in vivo. Protease digestion analysis suggested that Asp-Tyr could be the real effector of this penta-peptide in vivo.

Dioscorea batatas tuber lectin 1 (DB1) is a storage protein isolated from yam tuber, and is shown to be a mannose-binding lectin. It has 58% amino-acid identity to insecticidal snowdrop bulb lectin GNA. In this study, we demonstrated that ≥1 mg ml−1 DB1 in an artificial diet significantly decreased the survival and fecundity of green peach aphid, Myzus persicaeca. We produced transgenic tobacco plants expressing cDNA of DB1 under the control of Cauliflower mosaic virus 35S promoter (35S-DB1) or phloem-specific promoter of rice sucrose synthase-1 gene (RSs1-DB1), and evaluate the degree of aphid resistance in whole plant bioassays. The number of survival aphids was reduced to 60% in transgenic lines with 35S-DB1 and RSs1-DB1, which accumulated DB1 at a level of 1.8% and 0.25%, respectively, of total soluble protein. Our results indicate that DB1 can be used to enhance resistance to sap-sucking insects in transgenic crops.

A synthetic peptide related to locus adipokinetic hormone ( AKH ) and shrimp red pigment concentrating hormone ( RPCH ) containing a tyrosine residue in place of phenylalanine was iodinated and the 3,5- diiodotyrosyl derivative was isolated by reverse phase HPLC. Catalytic dehalogenation of the diiodo derivative in the presence of tritium yielded the tritiated AKH analog which was isolated by gel filtration on Sephadex LH-20 and reverse phase HPLC. The tritiated peptide was formed to be identical to AKH in its ability to stimulate lipid release into the hemolymph of locusts in vivo where the diiodotryrosyl derivative was inactive. The specific radioactivity of the tritiated peptide was 57.2 Ci/mmol, or 99% of the theoretical value.

Rice bran protein fraction (RBPF)—albumin, globulin, glutelin and prolamin were hydrolyzed with proteases M, N, P, S and pepsin under their optimal conditions for 24 h. Hydrolysates of various hydrolysis periods were collected and subjected to peptide mapping and the antioxidative activity measured by the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method. Protease M hydrolysates showed high degree of hydrolysis (DH), but low antioxidative activity. On the contrary, pepsin hydrolysates showed low DH with high activity. Albumin and globulin hydrolysates had higher DH values, but lower values for glutelin and prolamin. The globulin hydrolysate (Opep2) from 2 h-pepsin hydrolysis was separated by using three consecutive purification steps with RP-HPLC. Nineteen antioxidative peptides were isolated and their amino acid sequences were determined by a gas-phase protein sequencer and MALDI-TOF mass spectrometry. These peptides were composed of 6–30 amino acid residues with molecular masses ranging from 670–3,611 Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.

A rhamnose-binding lectin, named SFL, was isolated from the eggs of ayu (sweet fish, Plecoglossus altivelis) by affinity and ion-exchange chromatographies. SFL revealed 287 amino acid residues with 3 tandemly repeated domains, and contained 8 half-Cys residues in each domain. The lectin was shown to have a highly specific binding affinity to globotriaosylceramide (Gb3) by frontal affinity chromatography using 100 oligosaccharides. SFL was localized in several tissues and serum of both male and female ayu, such as gill, liver, ovary, testis, intestine, stomach, brain, kidney and serum. The lectin agglutinated the spores of the microsporidian Glugea plecoglossi, which is a pathogen of ayu. Although SFL bound to glycoproteins and glycolipids of G. plecoglossi spores, Gb3 could not be detected in either of them. The results suggest that SFL could interact with various glycoconjugates of pathogens to play a role in the adhesion of microorganisms invading in the body.

The effect of three plant lectins, soybean lectin (SBA), Japanese jack bean lectin (CGA), and wheat germ lectin (WGA), on the transport of various food factors, such as isoflavones, quercetin, dipeptides, and calcium ions, were investigated by use of an intestinal tract model, Caco-2 cell monolayers. The lectins increased the isoflavone transport but had no effect on aglycon transport. SBA increased the transport of quercetin glycosides, whereas CGA and WGA had no effect. The lectins increased the transport of calcium ions but showed no effect on the transport of dipeptides, carnosine, and anserine. Although SBA did not change the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayers, CGA and WGA decreased the TER value. These results indicate that plant lectins affect the transport of food factors in different manners, presumably due to their specific sugar binding activity.

A C-type lectin (BRA-2) isolated from the acorn barnacle Megabalanus rosa, which was a glycoprotein having an N-linked sugar chain, was deglycosylated by N-glycopeptidase F. The structure of the released sugar chains was determined by a 2-D mapping method after derivatization with a fluorescent reagent, 2-aminopyridine, to be Manα1–6(Manα1–3)Manβ1–4GlcNAcβ1–4(Fucα1–6)GlcNAc and Manα1–6(GlcNAcβ1–2Manα1–3)Manβ1–4GlcNAcβ1–4(Fucα1–6))GlcNAc. The structures were confirmed by matrix-assisted laser desorption ionization mass spectrometry and a comparison with authentic sugar chains by high-pressure liquid chromatography. Various properties of BRA-2 were examined before and after deglycosylation. The susceptibility of BRA-2 to protease digestion was increased by deglycosylation. However, the inhibitory activity toward calcium carbonate crystallization as well as the hemagglutinating activity of deglycosylated BRA-2 was significantly decreased. These results suggest that the sugar chains of BRA-2 are important to both its structural stability and its function.

L-amino acid oxidase (LAAO) activity, as well as mechanisms of antimicrobial action of aplysianin A, a sea hare-derived 340 kDa homotetrameric protein, was determined. Spectrophotometric and High-performance liquid chromatography analyses of aplysianin A indicated that one flavin adenine dinucleotide, a cofactor of LAAO, bound to each subunit of the homotetramer. Aplysianin A can specifically catalyze oxidation of basic amino acids (L-arginine and L-lysine), and is the first protein from marine invertebrate animals with LAAO activity. Substrate specificity of aplysianin A is markedly different from that of commonly known LAAO, such as snake venom LAAO, which prefer hydrophobic amino acids. Km value of aplysianin A was the smallest of those for all known LAAO reported. In the presence of catalase, the antibacterial activity of aplysianin A was inhibited as expected, indicating that the antibacterial action of aplysianin A results from hydrogen peroxide production during the reaction with substrates. Interestingly, aplysianin A acted as an antibacterial agent even in the presence of excess catalase. Antibacterial assays in various media suggested that this phenomenon was simply attributed to the consumption of amino acids required for bacterial growth in the media by aplysianin A.

Apios tuber lectin, named ATL, was isolated from Apios americana Medikus by two chromatography steps, hydrophobic chromatography and anion-exchange chromatography. The minimum concentration required for the hemagglutination activity toward rabbit erythrocytes of ATL was 4 μg/mL. ATL was composed of a homodimer of 28.4 kDa subunits. The amino acid sequence of ATL was similar to those of other legume lectins. The lectin showed moderate stability toward heating and acidic pH, and the binding affinity against several monosaccharides, such as D-glucosamine and D-galactosamine. ATL also bound to desialylated or agalactosylated glycoproteins such as asialo and agalacto transferrin. ATL decreased the transepithelial electrical resistance across human intestinal Caco-2 cell monolayers, suggesting the effect on the tight junction-mediated paracellular transport.

Peptidic fractions which inhibit angiotensin I-converting enzyme (ACE) were separated from peptic digests of soybean by ion exchange chromatography and gel filtration. Further separation of the peptidic fractions by ODS HPLC afforded active peptides, the amino acid sequences of which were identified by Edman 's procedure as: Ile-Ala (inhibitory against ACE with an IC 50 of 153 μM), Tyr-Leu-Ala-Gly-Asn-GIn (14 μM), Phe-Phe-Leu (37 μM), Ile-Tyr-Leu-Leu (42 μM), and Val-Met-Asp-Lys-Pro-Gln-Gly (39 μM). The antihypertensive activity of the soybean peptides was also investigated. Peptide fractions (2.0 g/kg body weight, oral administration) markedly lowered the blood pressure of spontaneously hypertensive rats (SHRs).

Seven kinds of angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from the hydrolysates of wakame (Undaria pinnatifida) by Protease S "Amano" (from Bacillus stearothermophilus) by using three-step high-performance liquid chromatography (HPLC) on a reverse-phase column. These peptides were identified by amino acid composition analysis, sequence analysis, and liquid chromatography-mass spectrometry (LC-MS), as Val-Tyr (IC(50) = 35.2 microM), Ile-Tyr (6.1 microM), Ala-Trp (18.8 microM), Phe-Tyr (42.3 microM), Val-Trp (3.3 microM), Ile-Trp (1.5 microM), and Leu-Trp (23.6 microM). These peptides have resistance against gastrointestinal proteases in vitro. Each peptide was determined to have an antihypertensive effect after a single oral administration in spontaneously hypertensive rats (SHR). Among them, the blood pressure significantly decreased by Val-Tyr, Ile-Tyr, Phe-Tyr, and Ile-Trp in a dose of 1 mg/kg of body weight (BW). The present study showed that antihypertensive effect in the hydrolysates of wakame by Protease S "Amano" was attributed to these peptides.

The amino acid sequence of a D-galactose-binding lectin isolated from the hemolymph of the acorn barnacle Balanus rostratus was determined. The lectin (BRL) (Mr 120K) is a multimeric protein whose subunit consists of 182 amino acids. The amino acid sequence was compared with those of multiple lectins (BRA-2, BRA-3) from Megabalanus rosa to explore the relationship between the structures and the inhibitory activity toward the crystal growth of calcium carbonate. Although BRL was 46% identical to BRA-2 and 15% identical to BRA-3, the lectin had no inhibitory activity, unlike BRA-2 and BRA-3. Both the number and the localization of acidic amino acid residues and their amide forms were different among them. Observations by scanning electron microscopy revealed modifications in the size and morphology of the calcium carbonate crystals grown in the presence of the lectins.

This study reports the isolation and partial characterisation of the ostrich serpin, alpha(2)AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich alpha(2)AP was purified using L-lysine-Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI-Sepharose chromatographies. It revealed a M(r) of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine alpha(2)AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human alpha(2)AP, DFP and EACA. Ostrich plasminogen was highly purified after L-lysine-Sepharose chromatography and showed a M(r) of 92 K, a total of 775 amino acids and its N-terminal sequence showed approximately 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a M(r) of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40 degrees C, respectively.

The amino-acid sequence of α-amylase isolated from the pancreas of the ostrich, Struthio camelus was determined. The α-amylase (OPA) consisted of 497 amino acid residues with pyroglutamic acid at the N-terminus and no oligosaccharide. Amino acid identity between OPA and chicken, porcine and human pancreatic α-amylases individually, was found to be 88, 82 and 86%, respectively.

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu2+-chelate Sepharose chromatography. Ostrich prothrombin exhibited a Mr of 72 800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin β-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins.

We have cloned and characterized the gene encoding Microcystis aeruginosa (strain M228) lectin (MAL). The gene contains 1551 nucleotides and an open reading frame for a protein of 517 amino acids with a predicted molecular weight of 55,159 Da. The carboxy-terminal region of MAL has three tandemly repeated homologous domains composed of 61 amino acids. These regions show similarity to the corresponding regions of the alpha-amylase of Clostridium beijerinckii (23% identity). The mal gene lies adjacent to an ORF that display homology to cytochrome P-450 and polyketide synthase. Southern hybridization showed that the genomic DNA of the strain M228 contained, in addition to MAL gene (mal), at least two other mal like gene.

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) was extracted from ostrich kidney cortex, intact kidney, ileum, pancreas, and liver. DPP IV was purified from the kidney cortex 373-fold by using homogenization, autolysis, diethylaminoethyl-Toyopearl 650 M, Cu 2+ -chelate affinity, and Mono Q chromatography. Autolysis conditions were investigated and optimized with regards to pH, incubation time, and Triton X-100 concentration. Nondenaturing gradient polyacrylamide gel electrophoresis (PAG) and sodium dodecyl sulfate-PAGE revealed molecular mass values of 270 K and 133 K for the native enzyme and its subunit, respectively. PAG-isoelectrofocusing indicated a pI of 4.7. The pH and temperature optimum were 8.0 and 45°C, respectively. The enthalpy of activation was computed as 20.91 kJ/mol. k cat and k cat / K m values were determined for various chromogenic substrates having the sequence of X-Pro-pNA. The effect of several metal ions on DPP IV activity was investigated. The N-terminus was blocked with an agent of unknown identity. The above data as well as the amino acid composition of ostrich DPP IV were compared with those of other mammalian and bacterial species. It appears that, in terms of physical characteristics, ostrich DPP IV correlated with those of other species, whereas chemically and kinetically it exhibited distinct characteristics.

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.

Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase–glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity chromatography, while jejunal sucrase–isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl–Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M r 145 000, 125 000 and 115 000. Although the N-terminal amino acid sequences bear no homology to SI, the M r 115 000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5. The glycohydrolases were most active at 58°C, but were quickly denatured above 60°C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters ( K m , k cat and k cat / K m ) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.

The properties of 22 synthetic peptides containing histidine, which were designed on the basis of the antioxidative peptide (Leu-Leu-Pro-His-His) derived from proteolytic digests of a soybean protein, were examined with regard to their antioxidative activity against the peroxidation of linoleic acid and the scavenging effects on active oxygen and free radical species. The antioxidative activities of these peptides in an emulsion oxidation system using 2,2'-azobis(2-amidinopropane) dihydrochloride as a radical initiator correlated well within an aqueous system. Although the histidine-containing peptides had a quenching activity on singlet oxygen, they did not show antioxidative activity in an 2,2'-azobis(2,4-dimethylvaleronitrile)-induced oxidation system or scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical and superoxide. The metal-ion chelating activities and the hydrophobicities of these peptides showed no direct correlation with their antioxidative activities. Leu-Leu-Pro-His-His was modified with a hydroxyl radical in an aqueous ethanol system during the peroxidation of linoleic acid.

Protease hydrolyses of a soybean protein, beta-conglycinin (7S protein), yielded antioxidative activity against the peroxidation of linoleic acid in an aqueous system at pH 7.0. Six antioxidative peptides were isolated from the hydrolysate prepared with protease S by size exclusion chromatography and reversed-phase HPLC. The amino acid sequences of the peptides were determined using a gas-phase protein sequencer and electron spray mass spectrometry. The peptides were composed of 5-16 amino acid residues, including hydrophobic amino acids, valine or leucine, at the N-terminal positions, and proline, histidine, or tyrosine in the sequences.

A fluorescent Edman-type reagent, fluorescein isothiocyanate, was adapted for gas-phase sequencing. Samples were covalently attached to functionalized polyvinylidene difluoride (PVDF) membranes or glass filter disks. In the case of sequencing the insulin B chain, the repetitive yields could be improved up to 98% by a covalent attachment of the peptide to an arylamine functionalized PVDF membrane from 74% for noncovalently immobilization on a PVDF membrane. The present method allowed the identification of 20 amino acid residues from the N-terminus with less than 5pmol of the insulin B chain.

An enzyme-linked immunosorbent assay for the multiple lectins (BRA-2, BRA-3) of the acorn barnacle Megabalanus rosa was developed. The assay was used to study the multiple lectin levels in the coelomic fluid and various tissue homogenates of M. rosa. The concentrations of the multiple lectins ranged from 0.13 to 2.2 mg/ml for BRA-2 and from 0.07 to 0.16 mg/ml for BRA-3, respectively. The protein concentration of the coelomic fluid ranged from 1.4 to 4.8 mg/ml. The multiple lectins in the coelomic fluid were also identified as the major proteins by two-dimensional gel electrophoresis.

A photoactivatable heterobifunctional fluorescent reagent, 1-azido-5-naphthalene sulfonyl (ANS) hydrazide, was synthesized and characterized. ANS-hydrazide reacted with lactose to form a photoactivatable hydrazone. The derivative (ANS-lactose) had the same binding affinity for a conger eel lectin as lactose judging from the hemagglutinating-inhibition assay with rabbit erythrocytes. ANS-lactose was used for photoaffinity labeling of a conger eel lectin. The photolabeled lectin was digested with chymotrypsin to isolate photolabeled peptides by reversed-phase HPLC by monitoring fluorescence. A major labeled peptide was located at positions 31-45 in the lectin by amino acid analysis and N-terminal sequencing. The identified segment was close to the highly conserved region throughout animal beta-galactoside-binding lectins.

The antifouling factor against the blue mussel Mytilus edulis found in the organic extract of the marine sponge Phyllospongia papyracea collected in Kagoshima was purified by solvent partition, silica gel column chromatography and HPLC on Toyopearl HW‐40 and on ODS. The active substance was analyzed by gas chromatography after methylesterification and identified as a mixture of several free fatty acids containing C16: 0 (59.9%), C16: l (11.8%) and C18: l (13.8%) as the major components. Among the various authentic free fatty acids tested, C10: 0, C14: l, C16: 0, C16.1, C18: 2, C20: 4 and fatty acids from porcine liver (FAPL) showed antifouling activity against M. edulis. The stronger activity of fatty acid mixtures compared with the same amount of each used singly suggested a synergistic effect of fatty acids on antifouling activity. FAPL inhibited larval settlement in the barnacle Balanus amphitrite without killing the larvae.

A photoactivatable D-galactosamine derivative was prepared by reaction of the amino group of D-galactosamine with 1-azide-5-naphthalene sulfonyl chloride (ANS-Cl). The derivative (GalN-ANS) inhibited the agglutination activity of an acorn barnacle lectin against rabbit erythrocytes to the same extent as D-galactosamine. We used GalN-ANS for photoaffinity labeling of the lectin. The photolabeled lectin was digested with pronase and the digest was separated by reversed-phase high-performance liquid chromatography by monitoring fluorescence and uv absorption to isolate the peptide labeled with GalN-ANS. Amino acid analyses of the labeled peptides revealed that GalN-ANS preferentially covalently labeled two regions in the carbohydrate recognition domain of the lectin. One of them was the highly conserved amino-acid sequence region throughout all calcium-dependent animal lectins.

Columns of phosphorylcholine (PC) immobilized on silica gel were shown to be useful for size exclusion chromatography (SEC) of proteins. The columns provided good separation of proteins in 50mM sodium phosphate buffer (pH 6.9) containing 0.25 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights with a correlation coefficient (R(2)) of 0.978-0.992. The columns were used in analyzing the subunit structures of the rhamnose-binding lectins CSL1, CSL2, and CSL3, isolated from chum salmon (Oncorhynchus keta) eggs. Although the lectins, which are a group of carbohydrate-binding and hydrophobic proteins, behaved anomalously in SEC with conventional matrices, they could be eluted from the immobilized PC columns without non-size-related retention, thereby allowing their molecular weights to be reliably estimated.

A myofibril-bound serine protease (MBSP) was partially purified from ostrich ( Struthio camelus ) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 °C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters ( K m and V max values) were calculated from Lineweaver–Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%).

The characterization of cod somatolactin (SL), a new pituitary protein belonging to the growth hormone/prolactin family, is described. Cod SL has a molecular weight of 26 kDa and consists of 209 amino acids, of which eight are Cys. The protein has three disulfide bonds between residues Cys5-Cys15, Cys65-Cys181, and Cys198-Cys206. The Cys residues at positions 42 and 180 are not involved in disulfide bonding. The positions of these disulfide bonds are homologous to those found in prolactin and growth hormone. Cod SL has two possible N-glycosylation sites, but only one appears to have carbohydrate units attached. Chemical analysis showed the following sugars to be present: galactose, mannose, N-acetylneuramic acid, and glucosamine. A smaller variant (23 kDa) of SL has been isolated, which is believed to be deglycosylated. Sequence comparison revealed cod SL to be similarly related to both GH and PRL, but slightly higher identity was observed to the tetrapod hormones (27-33%) than to the teleost hormones (21-27%).

A method was developed for analysis of sugar compositions of glycoproteins electroblotted onto polyvinylidene difluoride (PVDF) membranes after SDS-polyacrylamide gel electrophoresis. It was based on hydrolysis of glycoproteins on PVDF membranes and precolumn derivatization of liberated neutral sugars, sialic acids and aminosugars, respectively, and isocratic reversed-phase high-performance liquid chromatographies of these derivatives. The presence of sialo-glycoproteins in echinoderm shell matrixes was demonstrated by this method.

The addition of 3% (w/v) phenol to 6 M HCl largely prevented the destruction of tryptophan during rapid hydrolysis of peptides and proteins at 166 degrees C for 25 min or at 145 degrees C for 4 h. This hydrolysis procedure was advantageous for amino acid microanalysis using conventional high-performance liquid chromatography with a precolumn derivatization technique. The recovery of tryptophan from proteins was at least 80%. The addition of phenol also improved the recovery of methionine and carboxymethylcysteine. The amount of tryptophan in proteins electroblotted onto a polyvinylidene difluoride membrane was determined by this method.

The amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 140,000) is a multimeric protein whose subunit consists of 173 amino acids and one carbohydrate chain attached to Asn-39. The amino-acid sequence was determined by the manual sequencing of peptides derived from the protein by digestion with Staphylococcus aureus V8 proteinase, lysine endopeptidase and chymotrypsin, as well as fragments produced by cleavage with cyanogen bromide. The amino-acid sequence of the lectin was compared with the sequence of one (Mr 64,000) of the multiple lectins of M. rosa. They are distinct molecules in spite of a significant homology in their amino-acid sequences. The amino-acid sequence includes some regions homologous to those in other invertebrate lectins, such as sea urchin and flesh fly lectins, and vertebrate lectins. This is the first report to show the amino-acid sequence of multiple lectins isolated from an invertebrate.

The positions of the interchain and intrachain disulfide bonds and the glycosylation site in a lectin of the acorn barnacle Megabalanus rosa were determined. The lectin ( M r 140000 ) is composed of the same subunit ( M r 22000 ) which is cross-linked by disulfide bonds to form a dimer. Intact lectin yielded two fragments, CB1 and CB2, by cleavage with cyanogen bromide. One intrachain and two interchain disulfide bonds were identified as Cys-53-Cys-61, Cys-14-Cys-50′ and Cys-50-Cys-14′, respectively, by enzymatic digestion and Edman degradation of CB1. Two interchain disulfide bonds were determined as Cys-78-Cys-168 and Cys-144-Cys-160 by enzymatic digestion of CB2. The two interchain disulfide bonds are well conserved through all invertebrate lectins and calcium-dependent animal lectins. S- Carboxaminodomethylated lectin was digested with Staphylococcus aureus V8 proteinase and separated by reversed-phase HPLC. Glycopeptides were detected by the 4-N,N- dimethylamino -4′- azobenzene sulfonyl hyrazide method. Sequence analyses of the glycopeptides showed that a carbohydrate chain attached to Asn-39.

A method was developed for the analysis of reducing sugars extracted with 80% aqueous ethanol from fish muscle. It was based on the reaction of the reducing sugars with the chromo-phoric reagent, 4-N, N-dimethylamino-4'-azobenzene sulfonyl hydrazide, and their separation by reversed-phase high-performance liquid chromatography (HPLC) or high-performance silical gel thin-layer chromatography. The absorbance at 485nm provided a sensitivity of the picomole level. This method was applied to the reducing sugar analysis of 20mg of fish muscle. The change of reducing sugar contents in fish muscle of various stages of freshness was compared with that of K values measured by ion-pair reversed-phase HPLC.

In the present study, the entire amino acid sequence and cDNA structure encoding the d-galactose-binding lectin, SLL-2, isolated from the octocoral Sinularia lochmodes, were determined. SLL-2 regulates the morphology of symbiotic dinoflagellates Symbiodinium spp. through unknown mechanisms. Here, three cDNAs that encode SLL-2 were cloned and characterized. All the SLL-2 cDNAs encoded 142 amino acids with high similarity to each other. The mature subunit of SLL-2 was found to be composed of 94 amino acids and to contain one putative glycosylation site common to all three SLL-2. N-Glycopeptidase F treatment of SLL-2 resulted in a protein band shift from 16.5 to 9.5kDa in SDS-PAGE, confirming that SLL-2s are glycoproteins. Two-dimensional polyacrylamide gel electrophoresis analysis of the deglycosylated SLL-2 indicated a presence of three polypeptides as encoded in SLL-2 cDNAs. The deduced sequences of SLL-2 cDNAs had a similarity to the C-terminal region of discoidin I, the slime mold Dictyostelium discoideum lectin.

Autolysis of squid liver and mantle muscle homogenates, and blends of both, each yielded inhibitory activity toward the angiotensin I-converting enzyme. The inhibitory activity and the amount of solubilized protein in each of these autolysates were examined over a period of 24 h. Inhibitory peptides were isolated from the mixed autolysate, their structures (IC50) being Tyr-Ala-Leu-Pro-His-Ala (9.8 microM) and Gly-Tyr-Ala-Leu-Pro-His-Ala (27.3 microM).

Four major proteins designated DB1, DB2, DB3, and DB4 were isolated and characterized from the yam tuber Dioscorea batatas. The ratios of their yields were 20:50:20:10. DB1 was a mannose-binding lectin (20 kDa) consisting of 10-kDa subunits and was classified as the monocot mannose-binding lectin family. DB2, accounting for 50% of the total protein, was the storage protein, commonly called dioscorins consisting of a 31-kDa subunit. On the basis of amino acid sequence, DB2 was classified to be dioscorin A. DB3 was a maltose-binding lectin, having an apparent molecular mass of 120 kDa and composed of a 66-kDa subunit and two 31-kDa subunits (DB3S). The 66-kDa subunit was further composed of two 31-kDa subunits (DB3L) cross-linked by disulfide bonds. DB3L and DB3S (242 and 241 amino acid residues, respectively) were homologous with each other with 72% sequence identity. They showed a sequence homology to dioscorin B and dioscorin A from Dioscorea alata, with 90 and 93% identity, respectively, and to carbonic anhydrase from Arabidopsis thaliana with about 45% identity. DB3S had one intrachain disulfide bond located at Cys(28)-Cys(187), whereas DB3L had one interchain disulfide bond (Cys(40)-Cys(40)') in addition to the intrachain disulfide bond (Cys(28)-Cys(188)) to form a 66-kDa subunit. DB1 and DB3 agglutinated rabbit erythrocytes at 2.7 and 3.9 microg/ml, respectively. Despite the structural homology between DB2 and DB3, DB2 had no lectin activity. The 66-kDa subunit itself revealed the full hemagglutinating activity of DB3, indicating that DB3L but not DB3S was responsible for the activity. The hemagglutinating activity of DB3 required Ca(2+) ions and was exclusively inhibited by maltose and oligomaltoses (e.g. maltopentaose and maltohexaose) but not by d-glucose. DB3 could not be classified into any known plant lectin family. DB4 was a chitinase, homologous to an acidic chitinase from Dioscorea japonica. DB1, DB2, and DB3 did not show any activity of carbonic anhydrase, amylase, or trypsin inhibitor activity. These results show that two of the four major proteins isolated from the yam tubers D. batatas have unique lectin activities.

A kinetic investigation of ostrich thrombin specificity, its regulation and evolutionary development in comparison to those of other well-characterised species may contribute to the understanding of the structure-function relationships of thrombin. Antithrombin III (ATIII) was purified from ostrich plasma by heparin-Sepharose and Super Q-650S chromatography. It exhibited a M(r) of 59.2K and a pI in the range of 5.2-6.0. The ostrich N-terminal sequence was compared to those of other known species and showed the highest identity with rabbit ATIII (31%). Inhibition studies included the interaction of ostrich and human ATIII with bovine, human and ostrich thrombin. At a 2:1 molar ratio of ostrich ATIII to enzyme, 20 and 40% remaining activity was found for bovine and ostrich thrombin, respectively. Ostrich thrombin exhibited a pH and temperature optimum of 9.0 and 60 degrees C, respectively. Hydrolysis of seven peptide p-nitroanilide substrates by ostrich thrombin revealed D-Phe-Pip-Arg-pNA (k(cat)/K(m)=9.65 microM(-1)s(-1)) as the substrate with the highest catalytic efficiency. The effect of monovalent cations on ostrich thrombin catalysis revealed enhanced activity with Na(+). The calculated K(i) values for the complex formation between ostrich thrombin and ostrich (9.29 x 10(-11)M) and human (9.66 x 10(-11)M) ATIII are comparable to reported results. The results obtained from the present study confirmed that ostrich thrombin and ATIII are closely related to the corresponding molecules of other species in terms of physicochemical and kinetic properties.

Calcium-activated neutral proteinases (CANPs) and their endogenous specific inhibitor calpastatin are found in a wide variety of vertebrate and invertebrate tissues. The CANPs are cysteine proteinases that have an absolute requirement for Ca(2+) for activity. mu-Calpain and calpastatin were purified by successive chromatographic steps on Toyopearl-Super Q 650S and Pharmacia Mono Q HR 5/5 columns. The enzyme has a M(r) of 84KDa using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), a M(min) of 79KDa from amino acid analysis and an pI of 5.2. Calpastatin has a M(r) of 323KDa using denaturing gradient PAGE and a pI of 4.7. The amino acid composition of mu-calpain revealed 689 residues and the pH and temperature optima were found to be 7.5 and 37 degrees C, respectively. mu-Calpain underwent a Ca(2+)-dependent autoproteolysis producing a fragment of 82KDa. The N-terminal sequence of mu-calpain showed 24 and 18% sequence identity with human and bovine mu-calpain.

The proteasome is a high molecular weight, multisubunit and multicatalytic enzyme. Here we report the purification and characterization of ostrich skeletal muscle 20S proteasome. It was purified to homogeneity with Mr 700,000, pI 6.67 and a 'ladder' of 22.2-33.5 kDa bands on SDS-PAGE. The amino acid composition and amino-terminal sequences showed large identities to those of other species. For the three major activities, pH and temperature optima ranged between 8.0-11.0 and 40-70 degrees C, and stabilities between 5-12 and up to 40-60 degrees C. Substrate specificity and inhibitory effects were also studied. Many similarities to other sources were shown, with a few significant differences.

Protein hydrolysates, prepared by enzymatic digestion of soybean protein and egg white albumin using several proteases, inhibited the crystal growth of calcium carbonate. Each hydrolysate showed different inhibitory activities, suggesting the key role of peptide structures in the inhibition. The deamidation of protein hydrolysates by glutaminase increased not only the inhibitory activity toward the crystal growth of calcium carbonate but also the resistance of the hydrolysates against peptic digestion. Furthermore, the addition of sodium chloride, citric acid, or lactose into the reaction mixture enhanced the inhibitory activity. The protein hydrolysates inhibited both nucleation and crystal growth of calcium carbonate and also affected the crystal morphology.

Aeromonas caviae W-61 produces multiple extracellular xylanases, the xylanases 1, 2, 3, 4, and 5 [Nguyen, V. D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993)]. Here we purified and characterized high-molecular-weight xylanases, the xylanases 4 and 5 from the culture fluids of the bacterium. The purified xylanases 4 and 5, which had molecular masses of 120 and 140 kDa, respectively, were endo-beta-1,4-xylanases with similar enzymatic properties except for trans-xylosidase activity. The xylanase 4 showed a prominent transxylosidase activity when xylotriose and xylotetraose were used as the substrates, while the xylanase 5 had little transxylosidase activity under the same conditions. Protein sequencing indicated that the xylanase 4 was a C-terminally-truncated xylanase 5, suggesting that the C-terminal truncation of the xylanase 5 may endow the enzyme with transxylosidase activity.

The complete amino acid sequence of an isogalectin, named congerin II, isolated from the skin mucus of conger eel, was determined by sequencing of the protein and its peptides generated by enzymatic and chemical cleavages. Congerin II consisted of 135 amino acids residues containing an acetylated N-terminus. Congerin II was found to be only 46% homologous in sequence to congerin I which was previously determined (Muramoto K., Kamiya H., Biochem. Biophys. Acta, 1992;1116:129–136), suggesting that the galectins with diverse molecular properties are present in the skin mucus of conger eel. However, it was confirmed by analysis of the secondary structures using circular dichroism that both congerins I and II shared similar folds characterized by β structures. Congerins I and II showed different molecular properties such as thermostability, pH dependency for hemagglutinating activity and for binding specificity against the pyridylamino derivative of lactose. Congerin I showed more strict recognition specificity for lactose than did congerin II. Furthermore, the effects of chemical modification on congerins I and II were investigated in order to identify the type of amino acids involved in their different lectin activities. Modification of tyrosine and lysine residues did not affect the carbohydrate-binding activities of congerins. However, modification of tryptophan, arginine, histidine, glutamic acid and aspartic acid residues led to considerable loss of their activities, and a different mode of binding activity was observed between modified congerins I and II. These results suggest that multiple galectins from conger eel with the same scaffold have different biological functions and properties.

α 1 -Antichymotrypsin, a member of the serpins, is the predominant plasma inhibitor of neutrophil cathepsin G. The aim of this study was to purify ostrich α 1 -antichymotrypsin and to compare its biochemical properties with those of other species. Ostrich α 1 -antichymotrypsin was purified from serum by ammonium sulphate fractionation, QAE-Sephadex C-50 and phenyl-Toyopearl chromatography. N -terminal sequence, amino acid composition, molecular mass, isoelectric point and reaction with cathepsin G, elastase and chymotrypsin were determined. SDS-PAGE revealed a M r of 55 000 for ostrich α 1 -antichymotrypsin and pI values of 6.8 and 4.1–4.3 were obtained. The amino acid composition revealed 444 residues and the N -terminal sequence of the first 20 residues revealed a homology of 30% when compared with several other α 1 -antichymotrypsin sequences. Total inhibition of cathepsin G by ostrich α 1 -antichymotrypsin was found at a 4:1 molar ratio of inhibitor to enzyme which was similar to that found for commercial α 1 -antichymotrypsin. Immunological studies highlighted the lack of cross-reactivity between ostrich and human α 1 -antichymotrypsin. The study indicated that ostrich α 1 -antichymotrypsin-like molecule exhibited similar properties to human α 1 -antichymotrypsin although there were notable differences.

Protease hydrolyses of various proteins yielded antioxidative activity against the peroxidation of linoleic acid. From the hydrolysate of a soybean protein, six antioxidative peptides were isolated by size exclusion chromatography and reverse-phase high-pressure liquid chromatography (HPLC). The smallest peptide, Leu-Leu-Pro-His-His, of the above peptides was chosen as a model peptide to investigate the structure-activity relationship. A total of 28 structurally related peptides were synthesized and their antioxidative activities tested. His and Pro in sequence were found to play important roles in the antioxidative activity and, among the peptides tested, Pro-His-His was the most antioxidative. Antioxidative peptides showed synergistic effects with nonpeptidic antioxidants as observed in the peptide of the digests. The magnitude of the effects, however, did not correlate with the antioxidative activities of the peptides.