Peculiarities of substrate hydrolysis by endopeptidases from hepatopancreas of king crab

Kinetic parameters of hydrolysis of peptide and protein substrates by psychrophilic endopeptidases from hepatopancreas of the king crab Paralithodes camtschaticus (PC), in particular, by trypsin, collagenolytic protease, and metalloprotease, were measured at different temperatures. The PC trypsin was shown to hydrolyze Bz-Arg-pNA in the temperature range studied (4–37°C) 19 times more effectively than bovine trypsin. The rate constants of hydrolysis of Glp-Ala-Ala-Leu-pNA by the PC collagenolytic protease increased approximately by one order of magnitude along with temperature decrease, while K _m decreased by 3.5 times. The effective values of K _m for the hydrolysis of azocasein by the metalloprotease insignificantly depend on temperature. We proposed that electrostatic interactions of negative charges around the cavity of active site are critical for the effective hydrolysis of substrates by endopeptidases of the PC hepatopancreas.     

A collagenolytic serine protease with trypsin-like specificity from the fiddler crab Uca pugilator

A second collagenolytic serine protease has been isolated from the hepatopancreas of the fiddler crab, Uca pugilator. This enzyme cleaves the native triple helix of collagen under physiological conditions of pH, temperature, and ionic strength. In addition to its collagenolytic activity, the enzyme exhibits endopeptidase activity toward other polypeptides and small molecular weight synthetic substrates. The polypeptide bond specificity of this enzyme is similar to that of bovine trypsin as is its interaction with specific protease inhibitors. The amino-terminal sequence of this enzyme displays significant homology with other serine proteases, most notably with that of crayfish trypsin, and demonstrates that this enzyme is a member of the trypsin family of serine endopeptidases. The relatively unique action of this protease with regard to both collagenous and noncollagenous substrates has important implications concerning the specificity and mechanism of collagen degradation.     

Isolation of trypsin PC from the Kamchatka crab Paralithodes camtschatica and its properties]

Trypsin PC from the hepatopancreas of the king crab Paralithodes camtschatica was isolated and purified to apparent homogeneity by ion-exchange chromatography on Aminosilochrom and DEAE-Sephadex and affinity chromatography on arginine-agarose. The yield of the enzyme was 37.7%, and the purification degree was 21. Trypsin PC has a molecular mass of 29 kDa and pI < 2.5. It hydrolysis N-benzoyl-L-arginine p-nitroanilide at the optimum pH of 7.5-8.0 and at the temperature optimum of 55 degrees C (K(m) = 0.05 mM). Trypsin PC retained its activity within the pH range of 5.8-9.0 in the presence of Ca2+. The enzyme was inhibited by the specific inhibitors of serine proteases diisopropyl fluorophoshate and phenylmethylsulfonyl fluoride, by the trypsin inhibitor N-tosyl-L-lysylchloromethylketone, and by the trypsin inhibitors from soybean and potato. Trypsin PC was found to hydrolyze amide bonds formed by carboxylic groups of lysine and arginine in peptide substrates. The N-terminal sequence of this enzyme is IVGGTEVTPG.     

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.     

Triple-helical peptide analysis of collagenolytic protease activity

Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, non-fluorogenic and fluorogenic triple-helical peptide models of MMP-1 cleavage sites in interstitial collagens have been constructed. Triple-helical peptides were assembled by either (a) covalent branching or (b) self-association driven by hydrophobic interactions. Fluorogenic triple-helical peptide (fTHP) substrates contained the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5' positions, respectively. Investigation of MMP family hydrolysis of THPs showed kcat/Km values in the order of MMP-13 > MMP-1 approximately MMP-1(delta243-450) approximately MMP-2 > MMP-3. Studies on the effect of temperature on fTHP and an analogous fluorogenic single-stranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triple-helical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that 'triple-helical peptidase' activity can be distinguished from 'collagenolytic' activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.     

Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum.

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.     

The midgut trypsins of shrimp (Penaeus monodon). High efficiency toward native protein substrates including collagens

Two shrimp trypsins have been purified from the midguts of Penaeid shrimps by various chromatographies and HPLC. The molecular masses of them are 27 and 29 kDa, respectively. They show the typical specificity of trypsin for substrates and inhibitors, and their N-terminal amino-acid sequences are homologous to those of other trypsins. The shrimp enzymes are very acidic (pI less than or equal to 2.4), and show distinctively low Km for the synthetic amide substrates. They also hydrolyse various native proteins more efficiently than bovine trypsin in vitro. However, the anionic shrimp trypsins do not have special preference for basic protein substrates over the acidic one. Collagenolytic activity of the midgut extract was mainly due to serine proteases. The collagenolytic activity of the purified shrimp trypsin was confirmed by assays with either soluble or insoluble native type I collagens. In comparison with the other trypsins from the Crustacean decapods, the shrimp enzymes have four pairs of disulfide bonds, intermediary between the crayfish trypsin (three pairs) and the crab trypsin (five pairs), and are immunochemically different from them.     

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

Kinetic constants for the hydrolysis by porcine tissue beta-kallikrein B and by bovine trypsin of a number of peptides related to the sequence of kininogen (also one containing a P2 glycine residue instead of phenylalanine) and of a series of corresponding arginyl peptide esters with various apolar P2 residues have been determined under strictly comparative conditions. kcat and kcat/Km values for the hydrolysis of the Arg-Ser bonds of the peptides by trypsin are conspicuously high. kcat for the best of the peptide substrates, Ac-Phe-Arg-Ser-Val-NH2, even reaches kcat for the corresponding methyl ester, indicating rate-limiting deacylation also in the hydrolysis of a peptide bond by this enzyme. kcat/Km for the hydrolysis of the peptide esters with different nonpolar L-amino acids in P2 is remarkably constant (range 1.7), as it is for the pair of the above pentapeptides with P2 glycine or phenylalanine. kcat for the ester substrates varies fivefold, however, being greatest for the P2 glycine compounds. Obviously, an increased potential of a P2 residue for interactions with the enzyme lowers the rate of deacylation. In contrast to results obtained with chymotrypsin and pancreatic elastase, trypsin is well able to tolerate a P3 proline residue. In the hydrolysis of peptide esters, tissue kallikrein is definitely superior to trypsin. Conversely, peptide bonds are hydrolyzed less efficiently by tissue kallikrein and the acylation reaction is rate-limiting. The influence of the length of peptide substrates is similar in both enzymes and indicates an extension of the substrate recognition site from subsite S3 to at least S'3 of tissue kallikrein and the importance of a hydrogen bond between the P3 carbonyl group and Gly-216 of the enzymes. Tissue kallikrein also tolerates a P3 proline residue well. In sharp contrast to the behaviour of trypsin is the very strong influence of the P2 residue in tissue-kallikrein-catalyzed reactions. kcat/Km varies 75-fold in the series of the dipeptide esters with nonpolar L-amino acid residues in P2, a P2 glycine residue furnishing the worst and phenylalanine the best substrate, whereas this exchange in the pentapeptides changes kcat/Km as much as 730-fold. This behaviour, together with the high value of kcat/Km for Ac-Phe-Arg-OMe of 3.75 X 10(7) M-1 s-1, suggests rate-limiting binding (k1) in the hydrolysis of the best ester substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trypsin catalyzed hydrolysis of new chromogenic arginine substrates

Trypsin catalyzed hydrolysis of seven new chromogenic arginine substrates, N alpha-benzyloxycarbonyl-L-arginine-3-nitro-5X-anilide (X = H, CF3, SO2CH3, F, Cl, Br and I) were studied. These substrates are suitable for studying electronic effects on trypsin activity. The Km and kcat values for the hydrolysis of each substrate were determined and found to differ significantly for the various substrates. The Hammett plot of the catalytic rate constants gave a straight line with a negative rho value (-0.82) thus indicating that electron withdrawing substituents retard the trypsin catalyzed hydrolysis of the new anilide substrates.     

Aminopeptidase PC from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica

Homogeneous aminopeptidase PC was isolated with yield 67% and purification degree 237 from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica by ion-exchange chromatography on DEAE-Sepharose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephadex G-150. The enzyme is a homodimer with a molecular mass 220 kD (110 x 2). Aminopeptidase PC has pI = 4.1. It hydrolyzes Leu-pNA optimally at pH 6.0 and at the optimum temperature 36-40 degrees C; in the presence of Ca2+ the enzyme is stable at pH 5.5-8.0. Aminopeptidase PC is activated by Ca2+, Mg2+, and Fe2+; it is completely inhibited by EDTA, o-phenanthroline, and bestatin. The enzyme contains four Zn atoms per molecule and is therefore a metalloaminopeptidase. The aminopeptidase PC can effectively cleave N-terminal Arg and Lys residues as well as Leu, Phe, and Met residues. Km and kcat values for hydrolysis of Leu-pNA were 0.075 mM and 0.19 sec-1 and for hydrolysis of Arg-pNA 0.078 mM and 0.48 sec-1, respectively. D-Amino acid residues cannot be cleaved. Thus, aminopeptidase PC of the Kamchatka crab has a mixed substrate specificity which is characteristic of some microbe aminopeptidases. Its N-terminal sequence ESVEIELPEGLSPLV is 46% coincident with that of yeast vacuolar aminopeptidase YSCA.     

Kinetics of tryptic hydrolysis of the arginine-valine bond in folded and unfolded ribonuclease T1

We have used ribonuclease T1 and its chemically modified derivatives as substrates, and trypsin as proteinase, to investigate the kinetics of proteolysis of a specific peptide bond in the folded and unfolded conformations of a protein. Steady-state kinetic studies showed that Km = 0.27 mM and Kcat. = 2.45 s-1 for the tryptic hydrolysis of the Arg(77)-Val(78) peptide bond in unfolded ribonuclease T1. This Km is somewhat lower than, and the kcat. value similar to, values found for the tryptic hydrolysis of comparable bonds in small peptides. Our data for the initial velocity of hydrolysis of the Arg(77)-Val(78) bond in a solution of the folded protein indicate that the bond is at least 1700 times less rapidly hydrolysed in the folded than in the unfolded conformation of ribonuclease T1, and do not exclude the possibility that the bond is completely resistant to hydrolysis in the folded protein.     

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)     

Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein

We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.     

The allosteric effect of salt on human mast cell tryptase

The inhibitory effect of potassium chloride and ammonium sulphate on purified human skin tryptase and bovine trypsin was studied enzyme-kinetically, using Z-Gly-Pro-Arg-pNA, Z-Gly-Pro-Arg-AMC, benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME) as substrates. With increasing salt concentrations, the curve of reaction velocity vs. substrate concentration changed from hyperbolic to sigmoidal when anilide substrates (Z-Gly-Pro-Arg-pNA or -AMC) were used. Only the Km value increased, while the Vmax value remained unchanged. The trend was similar with BAEE or TAME as the substrates. However, the effect of salt on the hydrolysis of these ester substrates was not as strong as on the hydrolysis of anilide substrates, and sigmoidal kinetics were not observed even at the highest KCl concentration (0.7 M) used. Heparin, used as a stabilizer, had no influence on this phenomenon, but it did slightly decrease the apparent Km and Vmax values in low-salt conditions. By comparison, trypsin was not as strongly affected by salt as tryptase, and the inhibition type was mixed competitive and non-competitive. The present results indicate that the salt acts on tryptase as an allosteric effector, and this should be carefully considered when enzyme kinetic parameters and enzyme activity of skin tryptase are measured.     

A new beta-naphthylamide substrate of p-guanidino-L-phenylalanine for trypsin and related enzymes

N alpha-Benzyloxycarbonyl-p-guanidino-L-phenylalanine beta-naphthylamide (Z-GPA-beta NA) was synthesized and the susceptibility of this compound to trypsin and related enzymes was compared with that of N alpha-benzyloxycarbonyl-L-arginine beta-naphthylamide (Z-Arg-beta NA). Both Z-GPA-beta NA and Z-Arg-beta NA were rapidly and almost completely hydrolyzed by trypsin and pronase. Z-Arg-beta NA was hydrolyzed slowly by thrombin, while Z-GPA-beta NA was not susceptible to this enzyme at all. The rate of hydrolysis of Z-GPA-beta NA by papain was slower than that of Z-Arg-beta NA. Neither beta-naphthylamide substrate was hydrolyzed by alpha-chymotrypsin. The specificity constant (kcat/Km) for the hydrolysis of Z-GPA-beta NA by trypsin was somewhat larger than that for the hydrolysis of Z-Arg-beta NA. Contributions of the benzene ring in the side chain of Z-GPA-beta NA to good binding of this substrate to the specificity site of this enzyme and to the poor fit of the scissile bond in the substrate molecule to the active serine residue are presumed from comparison of the individual kinetic parameters (Km and kcat) for the two beta-naphthylamide substrates. Z-GPA-beta NA was ascertained to be a useful substrate in the study of the binding and catalytic specificities of various trypsin-like enzymes.     

Influence de la température sur quelques activités enzymatiques chez Palaemon serratus

Variation in Esterase 2 C activities, involving the hydrolysis of 2-carboxylic esters, α-glucosidase acetyl-glucosaminidase and alkaline and acid phosphotases, in the hepatopancreas and the abdominal muscle of Palaemon serratus was examined by polyacrylamide gradient gel electrophoresis. Soluble proteins were measured in the hepatopancreas and the abdominal muscle, and trypsin and chymotrypsin activities in the hepatopancreas. The activities and the isoenzymatic variations in shrimps acclimated at 5 different temperatures (between 14 and 30°) were compared and the molecular weight of each isozyme evaluated. It was found that: (a) the concentrations of soluble proteins decrease in the hepatopancreas between 18 and 30°, but remain unchanged in the abdominal muscle; (b) esterase and phosphatase activities increase with temperature but in a more or less random manner, according to the isozyme under consideration; (c) glycosidase activities increase with temperature; and (d) trypsin activity varies in an inverse relation to chymotrypsin activity.     

Substrate specificity of collagenolytic proteases from the hepatopancreas of the of the Kamchatka crab]

The substrate specificity of two isozymes of collagenolytic protease of the crab (Paralithodes camtschatica) was studied. It was found that both proteases can effectively hydrolyze type I and III collagens, as well as gelatin, the set of products yielded by enzymatic hydrolysis being different for isozymes A and C. Hydrolysis of some well-known peptides revealed that isozyme A predominantly cleaves the peptide bonds containing arginine and lysine residues, whereas isozyme C predominantly hydrolyzes bonds containing hydrophobic amino acids. The catalytic constants for the hydrolysis of several low molecular weight substrates in the presence of P. camtschatica proteases were determined, which allowed to attribute isozyme A to trypsin-like, and isozyme C to chymotrypsin-like proteinases. The peptide substrates of collagenase, Pz-Pro-Leu-Gly-Pro-D-Arg and Z-Gly-Pro-Ala-Gly-Pro-Ala are not hydrolyzed isozymes of crab collagenolytic protease.     

Phe5(4-nitro)-bradykinin: a chromogenic substrate for assay and kinetics of the metalloendopeptidase meprin

Phe5(4-nitro)-bradykinin has been identified as a good synthetic substrate to study the kinetics and mechanism of action of the metalloendopeptidase meprin. No convenient substrate for kinetic analysis of the enzyme had been previously described. HPLC analyses indicated that meprin cleaved bradykinin and nitrobradykinin between Phe5 (or Phe5(NO2)) and Ser6. Reaction rates for bradykinin were determined by quantitative HPLC analyses, whereas rates for nitrobradykinin were measured by continuous monitoring of the spectral change that occurs at 310 nm when the Phe(NO2)-Ser bond is hydrolyzed. For nitrobradykinin and unmodified bradykinin, respectively, Km values were 281 and 425 microM, kcat values were 28 and 22 s-1, and kcat/Km values were 9.7 x 10(4) and 5.1 x 10(4)M-1. The two products of bradykinin hydrolysis were not substrates for the enzyme, but they were inhibitors. The initial rates of hydrolysis of nitrobradykinin increased linearly with enzyme concentration (0.09-2.2 micrograms/ml), and increased linearly with temperature in the range from 15 to 55 degrees C. Hydrolysis of the substrate was optimal at alkaline pH values. The cysteine endopeptidases papain and cathepsin L and the metalloproteases thermolysin, angiotensin-converting enzyme, and neutral endopeptidase (EC 3.4.24.11) also cleaved nitrobradykinin, but at different peptide bonds than meprin. The single cleavage of nitrobradykinin at the Phe(NO2)-Ser bond and the concomitant spectral shift that occurs at alkaline pH makes this a particularly suitable substrate for meprin.     

Synthesis of fluorescent peptidyl thioneamides and the assay of papain in the presence of trypsin

Fluorescent peptidyl thioneamides are synthesized for the first time. The carbonyl oxygen of the scissile amide bond of the substrates was replaced by a sulfur atom. The proteolytic activities of trypsin and papain were measured against 5-(benzyloxycarbonyllysylthioamido)-isophthalic acid dimethyl ester (Z-Lys-psi[CS]-AIE) and 5-(benzyloxycarbonylphenylalanylarginylthioamido)-isophthalic++ + acid dimethyl ester (Z-Phe-Arg-psi[CS]-AIE) and were compared to the corresponding oxyamides. Kinetic constants were measured. With thioneamide substrates, no tryptic hydrolysis was observed. Papain, on the other hand, hydrolyzed both oxy and thioneamides. The Km values of the thioneamides were shown to be slightly lower for papain than for the oxyamides, but the efficiency of the overall catalytic activity was off set by the lower turnover number for the thio derivatives. With the present synthetic substrate technology, selective detection of cysteine proteases in the presence of serine proteases is difficult. The thioneamides reported here were hydrolyzed by papain alone in the presence of trypsin.     

A homologue of alpha 2-macroglobulin purified from the hemolymph of the horseshoe crab Limulus polyphemus

A high molecular weight protease inhibitor has been purified from the cell-free plasma of the horseshoe crab Limulus polyphemus using high speed centrifugation, polyethylene glycol precipitation, and gel filtration. The inhibitor is sensitive to mild acidification, methylamine treatment, and inhibits the proteolytic activity of a variety of endopeptidases. The molecule does not inhibit trypsin-mediated hydrolysis of low molecular weight substrates and protects the active site of trypsin from inactivation by soybean trypsin inhibitor. These properties are diagnostic of the alpha 2-macroglobulin (alpha 2M) class of protease inhibitors found in vertebrates. Like vertebrate alpha 2M the Limulus alpha 2M molecule is composed of subunits of molecular weight 180,000-185,000 as determined by polyacrylamide gel electrophoresis under reducing conditions. The apparent native molecular weight for the Limulus molecule as determined by both gel filtration and gel electrophoresis under nonreducing conditions is 500,000-550,000, compared to a native molecular weight of 700,000-750,000 for human alpha 2M, determined in parallel under identical conditions. These results suggest that alpha 2M appeared in evolution at least 550 million years ago before the divergence of the lineages that gave rise to present-day arthropods and mammals.