Biochemical characterization of trypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus)

A cationic trypsin (trypsin A) and an anionic trypsin (trypsin B) were highly purified from the hepatopancreas of the Japanese sea bass (Lateolabrax japonicus) by ammonium sulfate precipitation, column chromatographies of DEAE-Sepharose and Sephacryl S-200 HR. Purified trypsins revealed single band on SDS-PAGE and their molecular masses were 21 kDa and 21.5 kDa, respectively. Trypsins A and B exhibited maximal activity at 40°C, and shared the same optimal pH at 9.0 using Boc-Phe-Ser-Arg-MCA as the substrate. The two trypsins were stable up to 45°C and in the pH range from 7.0 to 11.0. Trypsin inhibitors such as Pefabloc SC, PMSF and benzamidine are effective to these two enzymes and their susceptibilities were similar. Apparent K(m)s of trypsins A and B were 1.12 and 0.7 μM and k(cat)s of them were 72.08 and 67.79 S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. The N-terminal amino acid sequences of the two trypsins were determined to the 24th residues, which were highly identical to trypsins from other species of fish while trypsins A and B only shared 45.8% identity. The digestive effect of the two trypsins on native shrimp muscular proteins indicated their effectiveness in the degradation of food proteins.     

Isolation and characterization of trypsin from pyloric caeca of Monterey sardine Sardinops sagax caerulea

Trypsin from pyloric caeca of Monterey sardine was purified by fractionation with ammonium sulfate, gel filtration, affinity and ionic exchange chromatography. Fraction 102, obtained from ionic exchange chromatography, generated one band in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing. The molecular mass of the isolated trypsin was 25 kDa and showed esterase-specific activity on Nalpha-p-tosyl-L-arginine methyl ester (TAME) that was 4.5 times greater than amidase-specific activity on N-benzoyl-L-arginine-p-nitroanilide. The purified enzyme was partially inhibited by the serine-protease phenyl-methyl-sulfonyl fluoride (PMSF) inhibitor and fully inhibited by the soybean trypsin inhibitor (SBTI) and benzamidine, but was not inhibited by the metallo-protease inactivator EDTA or the chymotrypsin inhibitor tosyl-L-phenylalanine chloromethyl-ketone. The optimum pH for activity was 8.0 and maximum stability was observed between pH 7 and 8. A marked loss in stability was observed below pH 4 and above pH 11. Activity was optimum at 50 degrees C and lost activity at higher temperatures. The kinetic trypsin constants K(m) and k(cat) were 0.051 mM and 2.12 s(-1), respectively, while the catalytic efficiency (k(cat)/K(m)) was 41 s(-1) mM(-1). General characteristics of the Monterey sardine trypsin resemble those of trypsins from other fish, especially trypsins from the anchovy Engraulis japonica and Engraulis encrasicholus and the sardine Sardinops melanostica.     

The two alpha 2-macroglobulin-bound trypsin molecules have different affinities for the basic pancreatic trypsin inhibitor

We have investigated the enzymatic properties of alpha 2-macroglobulin-bound porcine trypsin using a substrate: Z-Gly-Gly-Arg-p-nitroanilide and two inhibitors: p-aminobenzamidine and basic pancreatic trypsin inhibitor. The ternary alpha 2-macroglobulin-(trypsin)2 complex behaves like a mixture of two enzymes which bind basic pancreatic trypsin inhibitor with widely different affinities (Ki = 0.11 microM and 23 microM). About one-half of the trypsin molecules of the ternary complex are covalently bound to alpha 2-macroglobulin. Preparation of the complex in the presence of hydroxylamine prevents covalent bond formation, but the two trypsins of this artificial complex still exhibit large differences in affinity for basic pancreatic trypsin inhibitor. The trypsin molecules of the ternary complex also exhibit small differences in their affinity for Z-Gly-Gly-Arg-p-nitroanilide and p-aminobenzamidine.     

The Preparation of Trypsins and Chymotrypsins From Bovine and Porcine Residues After Insulin Extraction

Bovine and porcine pancreatic residue, remaining after the extraction of insulin, has been used to prepare a proteinase powder. This powder was used as a source of trypsin and chymo-trypsin. The individual enzymes were isolated and purified by chromatography on sulfopropyl (SP)-Sephadex C-25 and affinity chromatography on soybean trypsin inhibitor (STI)-Sepharose. The bovine proteinase powder contained a-chymotrypsin, trypsin and chymotrypsin B in the ratio 5:2:1. The porcine powder contained cationic trypsin, anionic trypsin and cationic chymotrypsin in the ratio 5 : 1. 4 : 3. The isolated enzymes were characterized and found to be identical with enzymes isolated from fresh tissue with the exception of porcine chymotrypsin. Porcine cationic chymotrypsin was isolated as two distinct forms, A-l and A-2, which appear to be different activation products of porcine chymotrypsinogen A. Both forms resemble bovine a-chymotrypsin, a three chain structure, rather than porcine chymo-trypsin A, a two chain structure. Furthermore, the B-chain appears to be cleaved, possibly at residues Phe₈₉-Lys₉₀.     

Interactions in vitro and in vivo between anionic and cationic porcine trypsin and porcine plasma proteinase inhibitors

A method for purifying porcine anionic and cationic trypsin is presented. Reaction mixtures with increasing amounts of the two porcine trypsins and porcine serum were studied in vitro to evaluate the relative importance of alpha 1-macroglobulin and alpha 2-macroglobulin as well as alpha 1-proteinase inhibitor in the rapid binding of porcine anionic and cationic trypsin. Porcine cationic trypsin was preferentially bound to alpha 1-macroglobulin, while anionic trypsin exhibited equal binding to both alpha-macroglobulins. Both trypsins were also bound by the alpha 1-proteinase inhibitor but not until alpha 1-macroglobulin approached saturation. Trypsin-alpha-macroglobulin complexes were cleared from plasma with a half-life of 6 min. For trypsin-alpha 1-proteinase inhibitor-complexes the half-life was 120 min. These findings are in accordance with results for other mammalian species, including man.     

Purification and characterization of trypsins from the digestive tract of Locusta migratoria

Two trypsin-like enzymes were isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides. Primary purification was carried out on a DEAE-cellulose column, from which the two trypsins emerged in the anionic fraction. Further purification was achieved by affinity chromatography on a p-aminobenzamidine (PABA)-Sepharose column, which also separated the two trypsins (TLEAff.1. and TLEAff.2.), or by HPLC on an anion exchange column. The purity and homogeneity of the trypsins were demonstrated by electrophoresis of cellulose acetate strips and in polyacrylamide gels, with and without SDS. The molecular weights of TLEAff.1 and TLEAff.2, as determined by SDS-PAGE, were 17,000 and 24,000 respectively. The amino acid compositions of the locust trypsins were similar to those of trypsins from the digestive systems of other insects, which are characterized by the lack or low content of half cystines. The isoelectric points were 3.2 for TLEAff.1 and 3.5 fold for TLEAff.2. Since most of the locust trypsin comprised TLEAff.2, the latter served as the main object of this study. TLEAff.2 was unstable at low pH, differing in this respect from mammalian trypsins. The optimum activity was at pH 8.5-9.0. The Km and kcat, values were similar to those for bovine trypsin. Activation by substrate, a phenomenon in bovine trypsin, was also observed for TLEAff.2. The locust trypsin was full inhibited by the proteinaceous trypsin inhibitors Bowman-Birk (BBI) and Kunitz from soybeans, CI from chickpeas, chicken ovomucoid (COM), and turkey ovomucoid (TOM). It was inactivated by phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK), indicating the involvement of serine and histidine in the active site.     

Kinetics of hydrolysis of amide and anilide substrates of p-guanidino-L-phenylalanine by bovine and porcine trypsins

The rates of hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalaninamide (Bz-GPA-NH2) and N alpha-substituted p-nitroanilides (pNA) of GPA (benzyloxycarbonyl(Z)-GPA-pNA, benzoyl(Bz)-GPA-pNA and acetyl(Ac)-GPA-pNA) by bovine and porcine trypsins were compared with those of arginine (Arg) substrates. The amide type substrates of GPA were hydrolyzed as fast as those of Arg by the two enzymes with much the same kcat/Km values, though significant differences were found between the kcat and Km values of GPA derivatives and those of Arg derivatives. The kinetic behavior of porcine trypsin toward GPA substrates was almost the same as that of the bovine enzyme. The ratio of the kcat value for Bz-GPA-OEt to that for Bz-GPA-NH2 was much larger than that for the ester to amide substrates of arginine, suggesting that the conformational change of the active site of trypsin induced by a benzene ring in the side chain of Bz-GPA-OEt specifically increases the velocity of the deacylation process of the ester substrate. Remarkably low values of both kcat and Km were found for the tryptic hydrolysis of Z-GPA-pNA and Ac-GPA-pNA, as well as on that of Bz-GPA-pNA (Tsunematsu, H., et al. (1983) J. Biochem. 94, 123-128). Z-GPA-pNA is the best substrate for the two trypsins among the three N alpha-substituted anilide substrates of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microscopic rate-constants for substrate binding and acylation in cold-adaptation of trypsin I from Atlantic cod

Temperature imposes limits on where life can thrive and this is evident in the evolution of the basic structural properties of proteins. Cold-adaptation of enzymes is one example, where the catalytic rate constant (k(cat)) is increased compared with hot-acclimated homologous under identical assay conditions. Trypsin I from Atlantic cod (Gadus morhua) has catalytic efficiency (k(cat)/K(m)) for amide hydrolysis that is 17-fold larger than observed for bovine trypsin. Here, the individual rate-constants for association of substrate (k(1)), dissociation of substrate (k(-1)), and acylation of the enzyme (k(2)) have been determined using benzoyl-Arg-p-nitroanilide or benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide as substrates. Rather unexpectedly, by far the largest difference (37-fold increase) was observed in k(1), the rate constant for binding of substrate. The cold-adaptation of the dissociation and catalytic steps were not as prominent (increased by 3.7-fold). The length of substrate did have an effect by increasing the reaction rate by 70-fold, and again, the step most affected was the initial binding-step.     

Use of natural dye-casein complexes: effect of proteolytic treatment

The activity of proteolytic enzymes is commonly measured using casein as a substrate. A modified caseinolysis assay was developed with natural dyes such as juglone, lawsone, berberine, and quercetin for Subtilisin carlsberg, protease type XVI, and trypsin, respectively. The pH dependence and incubation time were determined. K(m), V(max), and k(cat)/K(m) values were also determined for these enzymes. Lawsone was found to be a better substrate than the others.     

Purification and properties of rabbit trypsin.

The purification of rabbit pancreatic trypsin (EC 3.4.21.4) by affinity chromatography on Trasylol-Sepharose is presented along with its physical, chemical and immunological relationship to other trypsins. The molecule is a single polypeptide chain, which immunologically cross-reacts with porcine trypsin, but not with rabbit acrosomal proteinase. Sequence homology with other mammalian trypsins is seen at the amino terminus.     

Trypsins from yellowfin tuna (Thunnus albacores) spleen: purification and characterization

Two anionic trypsins (A and B) were purified to homogeneity from yellowfin tuna (Thunnus albacores) spleen by a series of column chromatographies including Sephacryl S-200, Sephadex G-50 and DEAE-cellulose. Purity was increased to 70.6- and 91.5-fold with approximately 2.8% and 15.6% yield for trypsin A and B, respectively. The apparent molecular weight of both trypsins was estimated to be 24 kDa by size exclusion chromatography and SDS-PAGE. Both trypsin A and B appeared as a single band on native-PAGE. Trypsin A and B exhibited the maximal activity at 55 and 65 degrees C, respectively, and had the same optimal pH at 8.5 using TAME as a substrate. Both trypsins were stable to heat treatment up to 50 degrees C and in the pH range of 6.0 to 11.0. Both trypsin A and B were stabilized by calcium ion. The activities were inhibited effectively by soybean trypsin inhibitor, TLCK and partially inhibited by EDTA, but were not inhibited by E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A. Activity of both trypsins continuously decreased with increasing NaCl concentration (0-30%). Apparent Km and Kcat of trypsin A and B for TAME were 0.2-0.33 mM and 66.7-80 S(-1), respectively. The N-terminal amino acid sequences of trypsin A, IVGGYECQAHSQPHQVSLNA, and trypsin B, IVGGYECQAHSQPPQVSLNA, indicated the high homology between both enzymes.     

Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting

Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the alpha subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).     

Purification,properties and substrate specificity of a digestive trypsin from Periplaneta americana (Dictyoptera) adults

A digestive trypsin from the American cockroach (Periplaneta americana, Dictyoptera) males was purified by a combination of anionic chromatographies in low and high pressure systems. The yield was 70% with a final specific activity of 2,000 units per mg protein (substrate: benzoyl-Arg-p-nitroanilide, BRpNA). Chemical modification with TLCK (k(obs)=3.3 M(-1) s(-1); stoichiometry 1:1) and PMSF (k(obs)=0.18 M(-1) s(-1); stoichiometry 1:1) confirmed that this peptidase is a trypsin. This enzyme has a molecular weight of 29 kDa (SDS-PAGE), a pI of 6.0 and a pH optimum of 8.9. Kinetic parameters using different colorimetric, fluorimetric and internally-quenched substrates indicated that P. americana trypsin prefers to hydrolyze synthetic substrates containing more than one amino acid residue and with an arginine residue at P1 position and a hydrophobic residue at P2. This enzyme presented a Km of 120 microM for BRpNA and is competitively inhibited by benzamidine (Ki=0.25 microM). Soybean trypsin inhibitor is a tight-binding inhibitor presenting a K(D) of 0.4 nM. Differences in substrate specificity and in the reactivity of the trypsin active site groups can be related to adaptation of insects to different hosts. P. americana trypsin is an excellent model for comparison as a basal group on evolutionary studies of insect trypsins.     

Electrostatics of mesophilic and psychrophilic trypsin isoenzymes: qualitative evaluation of electrostatic differences at the substrate binding site

A qualitative evaluation of electrostatic features of the substrate binding region of seven isoenzymes of trypsin has been performed by using the continuum electrostatic model for the solution of the Poisson-Boltzmann equation. The sources of the electrostatic differences among the trypsins have been sought by comparative calculations on selective charges: all charges, conserved charges, partial charges, unique cold trypsin charges, and a number of charge mutations. As expected, most of the negative potential at the S(1) region of all trypsins is generated from Asp(189), but the potential varies significantly among the seven trypsin isoenzymes. The three cold active enzymes included in this study possess a notably lower potential at and around the S(1)-pocket compared with the warm active counterparts; this finding may be the main contribution to the increased binding affinity. The source of the differences are nonconserved charged residues outside the specificity pocket, producing electric fields at the S(1)-pocket that are different in both sign and magnitude. The surface charges of the mesophilic trypsins generally induce the S(1) pocket positively, whereas surface charges of the cold trypsins produce a negative electric field of this region. Calculations on mutants, where charged amino acids were substituted between the trypsins, showed that mutations in Loop2 (residues 221B and 224) and residue 175, in particular, were responsible for the low potential of the cold enzymes.     

Histidine mapping of serine protease: a synergic study by IMAC and molecular modelling

The immobilized metal ion affinity (IMA) interaction of different serine proteases, namely porcine and bovine trypsins and BPN' and Carlsberg subtilisins, was studied on Sepharose-IDA-CuII. Both trypsins were resolved into their different subspecies, whereas the subtilisins appeared as only one species. The use of diethyl pyrocarbonate-modified enzymes demonstrated the contribution of histidine(s) as the sole interacting site(s). The use of different peptidic and chemical inhibitors complexed to the enzymes confirmed the contribution of histidine(s) as the interacting site(s) and further resulted in different chromatographic patterns for the free and complexed serine proteases. Comparison of the chromatographic data for each enzyme with the accessible surface area calculation by molecular modelling on the available crystallographic structure allowed us to hypothesize a map of the surface-accessible histidine on each enzyme.     

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

Properties of lactate dehydrogenase from the isopod, Saduria entomon

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.     

Sugar esterification catalysed by alkylated trypsin in dimethylformamide

Summary Reductive alkylation of porcine pancreatic trypsin with acetaldehyde, propionaldehyde, octaldehyde and benzaldehyde resulted in about 5 to 6 folds increase in the sugar esterification activities of the enzyme in DMF. The optimum activities of the modified enzymes depend on the degrees of their modification with the respective aldehydes. These alkylated trypsins were more stable in DMF compared to the native unmodified enzyme at temperatures between 26 and 60°C.     

Polyol-specific long-chain dehydrogenases/reductases of mannitol metabolism in Aspergillus fumigatus: biochemical characterization and pH studies of mannitol 2-dehydrogenase and mannitol-1-phosphate 5-dehydrogenase

Functional genomics data suggests that the metabolism of mannitol in the human pathogen Aspergillus fumigatus involves the action of two polyol-specific long-chain dehydrogenases/reductases, mannitol-1-phosphate 5-dehydrogenase (M1PDH) and mannitol 2-dehydrogenase (M2DH). The gene encoding the putative M2DH was expressed in Escherichia coli, and the purified recombinant protein was characterized biochemically. The predicted enzymatic function of a NAD(+)-dependent M2DH was confirmed. The enzyme is a monomer of 58kDa in solution and does not require metals for activity. pH profiles for M2DH and the previously isolated M1PDH were recorded in the pH range 6.0-10.0 for the oxidative and reductive direction of the reactions under conditions where substrate was limiting (k(cat)/K) or saturating (k(cat)). The pH-dependence of logk(cat) was usually different from that of log(k(cat)/K), suggesting that more than one step of the enzymatic mechanism was affected by changes in pH. The greater complexity of the pH profiles of log(k(cat)/K) for the fungal enzymes as compared to the analogous pH profiles for M2DH from Pseudomonas fluorescens may reflect sequence changes in vicinity of the conserved catalytic lysine.