Hydrolysis Of Tosyl Arginine Methyl Ester Research Articles

The pH Dependence of Serpin-Proteinase Complex Dissociation Reveals a Mechanism of Complex Stabilization Involving Inactive and Active Conformational States of the Proteinase Which Are Perturbable by Calcium

Serpin family protein proteinase inhibitors trap proteinases at the acyl-intermediate stage of cleavage of the serpin as a proteinase substrate by undergoing a dramatic conformational change, which is thought to distort the proteinase active site and slow deacylation. To investigate the extent to which proteinase catalytic function is defective in the serpin-proteinase complex, we compared the pH dependence of dissociation of several serpin-proteinase acyl-complexes with that of normal guanidinobenzoyl-proteinase acyl-intermediate complexes. Whereas the apparent rate constant for dissociation of guanidinobenzoyl-proteinase complexes (k(diss, app)) showed a pH dependence characteristic of His-57 catalysis of complex deacylation, the pH dependence of k(diss, app) for the serpin-proteinase complexes showed no evidence for His-57 involvement in complex deacylation and was instead characteristic of a hydroxide-mediated deacylation similar to that observed for the hydrolysis of tosylarginine methyl ester. Hydroxylamine enhanced the rate of serpin-proteinase complex dissociation but with a rate constant for nucleophilic attack on the acyl bond several orders of magnitude slower than that of hydroxide, implying limited accessibility of the acyl bond in the complex. The addition of 10-100 mm Ca(2+) ions stimulated up to 80-fold the dissociation rate constant of several serpin-trypsin complexes in a saturable manner at neutral pH and altered the pH dependence to a pattern characteristic of His-57-catalyzed complex deacylation. These results support a mechanism of kinetic stabilization of serpin-proteinase complexes wherein the complex is trapped as an acyl-intermediate by a serpin conformational change-induced inactivation of the proteinase catalytic function, but suggest that the inactive proteinase conformation in the complex is in equilibrium with an active proteinase conformation that can be stabilized by the preferential binding of an allosteric ligand such as Ca(2+).

CHARACTERIZATION OF TRYPSIN PURIFIED FROM THE PYLORIC CAECA OF THE SOUTHWEST ATLANTIC WHITE CROAKER Micropogonias furnieri (Sciaenidae)

A southwest Atlantic croaker protease was purified from the pyloric caeca by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The enzyme was classified as a trypsin on the basis of molecular weight, its ability to hydrolyze synthetic substrates N-α-benzoyl-arginine-p-nitroanilide (BAPA) and tosylarginine methyl ester (TAME), and its inhibition by known trypsin inhibitors. The isolated enzyme has a single band on SDS-PAGE with an estimated molecular mass of 24 kDa. Croaker trypsin activity was stable between pH 5 and pH 11 for 30 min at 0C, 10C and 25C and its maximal activity against BAPA was at pH 9.5. Thermostability was observed to about 55C for 30 min at pH 7.8 and its temperature optimum was 60C. Substrate turnover number was 850 BAPA units per μmol trypsin, and the K m was 0.081 mM at 25C. For the hydrolysis of TAME, V max was 9273 units per μmol trypsin and the K m was 0.155 mM at 25C. The catalytic efficiency V max /K m was higher than that of trypsin from fish living in colder waters.

KINETIC AND THERMODYNAMIC CHARACTERISTICS OF A DIGESTIVE PROTEASE FROM ATLANTIC COD, GADUS MORHUA

A 23,500 dalton protease was isolated from the pyloric ceca of Atlantic cod by the successive steps of ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The protein fraction recovered after affinity chromatography migrated as one band in both Davis and Laemmli gels. The protease was classified as trypsin (EC 3.4.21.4) on the basis of its substrate specificity, molecular weight and response to known trypsin inhibitors. For trypsin hydrolysis of benzoyl-DL-arginine p-nitroanilide, the substrate turnover number was 250 BAPA units per micromole trypsin (25°C), Km1 was 1.48 mM, the Arrhenius energy of activation (Ea) was 8.9 kcal/mol, and the free energy of activation (ΔG*) was 12.8 kcal/mol. The Vmax for the hydrolysis of tosylarginine methyl ester was 18,210 TAME units per micromole trypsin and the Km1 for the same reaction was 0.22 mM at 25°C.

Purification and characterization of trypsin from the Greenland cod (Gadus ogac). 1. Kinetic and thermodynamic characteristics

Trypsinogen was isolated from the pyloric ceca of Greenland cod by ammonium sulfate fractionation followed by acetone precipitation, and the trypsin(ogen) thus obtained was purified by affinity chromatography on soybean trypsin inhibitor – Sepharose 4B. The purified trypsin migrated as a single zone during polyacrylamide gel electrophoresis and its identity as trypsin (EC 3,4.21.4) was established by its catalytic specificity for amide or ester bonds involving the carboxyl group of arginine, its sensitivity to serine protease inhibitors and soybean trypsin inhibitor, and its molecular weight of 23 500. With tosylarginine methyl ester (TAME) as substrate, the turnover number of the hydrolytic reaction was about three times greater for the cod trypsin than for bovine trypsin at 5 °C. The Michaelis–Menten constant (Km,app) for cod trypsin and TAME increased from 0.14 mM at 5 °C to 0.26 mM at 35 °C, while the Km,app for bovine trypsin – TAME was about 0.05 mM at all assay temperatures. The free energy of activation (ΔG*) for the hydrolysis of TAME was about 600 cal/mol (1 cal = 4.1868 J) lower for the cod trypsin than for bovine trypsin at 5 °C. The contribution of enthalpy of activation (ΔH*) and entropy of activation (ΔS*) to ΔG* differed considerably for the two enzymes. The "physiological efficiency" (Vmax/Km,app) of the two enzymes with TAME was similar at 5 °C, but was much greater for bovine trypsin than cod trypsin at warmer temperatures. With N-α-benzoylarginine-p-nitroanilide (BAPA) as substrate, the turnover number was about eight times greater for the cod trypsin at 25 °C. The Km,app for cod trypsin – BAPA increased from 1.67 mM at 25 °C to 1.84 mM at 35 °C, whereas the Km,app for bovine trypsin – BAPA decreased from 0.97 mM at 25 °C to 0.90 mM at 35 °C. The ΔG* for hydrolysis of BAPA was about 1800 cal/mol lower for cod trypsin than it was for bovine trypsin at 25 °C. Vmax/Km,app was three to four times greater for cod trypsin than for bovine trypsin at 25 and 35 °C. These results show that Greenland cod trypsin is a better catalyst than bovine trypsin at low temperatures and that catalysis by the fish trypsin is less responsive to temperature change than is catalysis by bovine trypsin.

Effect of an extract of nereis virens on mammalian spermatozoa

AbstractEpididymal sperm of the mouse, rat, and guinea pig and ejaculated sperm of rabbits are cleaved at the head‐tail junction by an extract of Nereis virens. Annelids are extracted with water and the extract is purified by ion exchange chromatography. Electron microscopy shows that the extract acts on the filaments connecting the capitulum of the tail with the basal plate lining the nuclear envelope. Following detachment, the basal plate remains with the head. The extract contains proteases as indicated by hydrolysis of tosyl arginine methyl ester (TAME), benzoyl arginine ethyl ester (BAEE), and Azocoll, a general protease substrate. The hydrolysis of TAME is inhibited by tosyl lysine chloromethyl ketone (TLCK), a trypsin inhibitor, but TLCK does not prevent head‐tail separation by the Nereis extract. Similarly tosyl phenylalanine chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and phosphoramidon and leucyltryptophan, both thermolysin and acrolysin inhibitors — singly or in combination — do not prevent hydrolysis of Azocoll. Head‐tail separation activity of the extract was inhibited by dithiothreitol, which reduces disulfide bonds, and phenylmethyl sulfonyl fluoride, an inhibitor of serine proteases. These results indicate that the extract is a mixture of proteases, one being a serine protease similar to trypsin.Digestion of the connecting filaments with the pure proteases, trypsin and Staphylococcus aureus V8 protease, has yielded the following information on the proteins of the filaments. The accessibility of arginine and/or lysine peptide bonds to enzyme action is highest in rat sperm filaments, whereas those in the filaments of mouse, rabbit, and guinea pig sperm are less accessible than in the rat. Another possibility is that the total content of arginine and/or lysine varies between the species. The most dramatic difference is the enzymatic action on glutamyl peptide bonds of the filaments, the order being: mouse 〉 rat 〉 rabbit, with guinea pig sperm filaments completely resistant over the time course of the experiment.

Effects of SK-potentiator and fibrinogen on SK-plasminogen complex in the presence of tranexamic acid.

Abstract The addition of tranexamic acid to the mixture of plasminogen and streptokinase (called SK-activator activity) resulted in decreased extent of hydrolysis of TAMe (tosyl arginine methyl ester). The addition of SK-potentiator to the mixture of SK (streptokinase), plasminogen and tranexamic acid prevented the decrease in SK-activator activity caused by tranexamic acid, thus SK-potentiator counteracting with the effects of tranexamic acid. Fibrinogen potentiated SK-activator activity, but did not prevent the decrease of the activity caused by tranexamic acid. Fibrinogen added to SK and plasminogen prior to tranexamic acid prevented the decrease in SK-activator activity. From these data it is suggested that SK-potentiator and fibrinogen bind with lysine binding sites of plasminogen part of SK-activator, and SK-potentiator binds with SK-plasminogen (or -plasmin) complex faster than fibrinogen.

Hydrolysis of chromogenic substrates and tame by the mixture of α2macroglobulin and trypsin

When trypsin was added to plasma, hydrolysis of fluorescamine-labeled casein (f-casein) was inhibited, but hydrolysis of TAMe (tosyl arginine methyl ester) was rather enhanced. The hydrolysis of f-casein by the mixture of α 2M and trypsin was inhibited, however a slight, but still significant extent of hydrolysis was observed. The hydrolysis of TAMe by α 2M and trypsin was higher than trypsin itself, but that of S-2222 by α 2M-trypsin complex was less than trypsin itself. Comparison of the extent of hydrolysis of various substrates by α 2M-trypsin complex shows that some substrates (S-2222, S-2444, S-2160, S-2238) were less hydrolyzed by α 2M-trypsin complex than by trypsin, but some substrates (TAMe, S-2251) were more hydrolyzed. The extent of hydrolysis of various substrates by α 2M-trypsin complex was not merely determined by molecular weights of these substrates, nor sensitivity of these substrates to trypsin.

Inhibition of bovine trypsin with human plasma inhibitors

Hydrolysis of TAMe (tosyl arginine methyl ester) by trypsin was not inhibited by undiluted plasma or its pseudoglobulin fraction. Dilution of plasma resulted in inhibition of trypsin activity. Inhibition of tryptic hydrolysis of TAMe by urinary trypsin inhibitor (UTI) was prevented by macroglobulin fractions of pseudoglobulin. When case in was used as the substrate a similar phenomenon was not observed because the trypsin- α 2macroglobulin complex does not hydrolyze casein. Highly purified α 2macroglobulin ( α 2M) protected trypsin against other proteinase inhibitors ( α 1antitrypsin or UTI). Mixture of 1.1 μM of α 1antitrypsin ( α 1AT) and 9.1 × 10 −2μM of α 2M did not inhibit 8.3 × 10 −2μM of trypsin significantly, but one tenth amounts of α 1AT (1.1 × 10 −1μM) and 9.1 × 10 −3μM of α 2M showed a large extent of inhibition of 8.3 × 10 −2μM of trypsin. These results may suggest that α 2M rather protected trypsin from its inactivation by other proteinase inhibitors. Such α 2M-trypsin complex still hydrolyzes molecules of certain molecular weights such as tripeptide-pNA (H-D-Val-Leu-Lys-pNA, M.W. 552).

α-Keto derivatives of amino acids as inhibitors of trypsinogen activation

Deaminated analogues of tyrosine, tryptophan, phenylalanine, and glutamic acid were found to possess a significant inhibitory effect on the autocatalytic activation of trypsinogen and also on the activation by enterokinase. The α-keto derivatives of the three aromatic amino acids were the strongest inhibitors in the group, and of these, p-hydroxyphenylpyruvic acid was the most active compound. In its ability to block both types of activation of trypsinogen, p-hydroxyphenylpyruvic acid was superior to γ-guanidinobutyric acid, the most effective inhibitor previously described. The inhibition of enterokinase was noncompetitive for p-hydroxyphenylpyruvic acid and phenylpyruvic acid, but was competitive for indolepyruvic acid. In the autocatalytic reaction all three aromatic keto acids as well as γ-guanidinobutyric acid behaved as noncompetitive inhibitors. At a concentration of 10 −3 M the aromatic amino acids did not interfere with the caseinolytic activity of trypsin, though this concentration was sufficient to markedly inhibit the autocatalytic process. At the same concentration only indolepyruvic acid showed mild inhibition of the tryptic hydrolysis of tosyl-arginine methyl ester, and the kinetics were those of a noncompetitive reaction.

The action of thrombin on serum albumin

In 1951, Bailey et al published the first direct evidence that thrombin acts as a protease in the conversion of fibrinogen to fibrin. Bettleheim and Bailey (1952) showed subsequently that thrombin catalyzes the hydrolysis of two peptides from the fibrinogen molecule. In addition, thrombin has been reported to attack casein ( Schultz and Schwick, 1951) and probably gelatin ( Kay, 1951). Guest and Ware (1950) found that thrombin, at a concentration 10,000 times that needed for coagulation, would dissolve fibrin. The lysis of fibrin by thrombin has been confirmed by Seegers and coworkers (1958) who used thrombin with the highest activity yet obtained, 4100 units per mg. Bailey and Bettleheim (1955) reported no effect on rabbit myosin or ovalbumin. Sherry and Troll (1954) have shown that thrombin catalyzes the hydrolysis of tosylarginine methyl ester. It has also been reported to hydrolyze lysine ethyl ester slowly ( Ehrenpreis et al, 1957). It appears that thrombin has a limited proteolytic activity with substrate requirements similar to trypsin but more specific. The present report showing a limited digestion of albumin by thrombin is part of a study to determine the possible usefulness of thrombin in studies of protein structure.

Nature of the thrombin-inhibiting effect of incubated fibrinogen.

This study investigated the effect of a number of factors on the inhibition of thrombin by the anticoagulant fraction of incubated fibrinogen (AFIF). Heating above 46°C reduces and at 55°C completely abolishes the thrombin-inhibiting activity of AFIF. The prolongation of the thrombin time of fibrinogen by AFIF is proportional to the pH of the reaction mixture, while that of plasma tends to level off beyond pH 7.5. Calcium and magnesium up to a concentration of 10 mm shorten the thrombin time of both plain fibrinogen and fibrinogen mixed with AFIF in a proportional manner. AFIF partially inhibits the thrombin time reducing property of protamine on fibrinogen. Related probably to this is the observation that AFIF and serum protect purified fibrinogen from precipitation by protamine. The inhibitory effect of AFIF on the action of thrombin on fibrinogen was found to be competitive in nature. The hydrolysis of tosylarginine methylester by thrombin was shown to be unaffected by AFIF. The properties of the anticoagulant studied did not allow its identification with heparin.