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PubMed1997

The involvement of a novel mechanism distinct from the thrombin receptor in the vasocontraction induced by trypsin.

1. The vasocontracting effect of a serine protease trypsin and its mechanisms were investigated by monitoring the isometric tension in endothelium-denuded rings of rabbit thoracic aortae and its effects on intracellular free Ca2+ concentrations ([Ca2+]i) in dispersed rabbit vascular smooth muscle cells with a Ca2+ indicator fura-2. The actions of trypsin were compared with those of thrombin. 2. Both thrombin and trypsin reversibly contracted aortic rings without endothelium in a concentration-dependent manner. The vasocontraction induced by trypsin was well correlated with the protease activity of trypsin actually added to the tissue baths containing the aortic rings and was completely blocked by soybean trypsin inhibitor and phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor. 3. The trypsin-induced contractions of the aortic rings were not the result of irreversible damage to vascular smooth muscle cells, since the contractile responses induced by noradrenaline or 30 mM KCl were unaffected by pretreatment with trypsin. 4. The contractions induced by either thrombin or trypsin were reduced to about 30% of control responses after removal of extracellular Ca2+, indicating that most of the contraction is dependent on extracellular Ca2+. By contrast, the contractions induced by either of the proteases were reduced by an antagonist of L-type voltage-operated Ca2+ channels, nifedipine, to about 70% of control responses, indicating that both nifedipine-sensitive and -resistant Ca2+ channels are involved in these contractions. 5. In the aortic rings precontracted by a maximally effective concentration of thrombin, the second application of thrombin virtually failed to induce contractions but trypsin could still induce contractions amounting to 10% of control values by it's protease activity. 6. After the first application of a maximal concentration of thrombin, the second application of thrombin could not induce an increase in [Ca2+]i, but an application of trypsin could still induce an increase in [Ca2+]i in dispersed rabbit vascular smooth muscle cells. 7. These data suggest that in addition to activation of a thrombin receptor, trypsin can contract rabbit aortae by a proteinase-activated receptor 2 or a novel mechanism.

Animals↗
PubMed2001

Mechanism of trypsin-induced contraction in the rat myometrium: the possible involvement of a novel member of protease-activated receptor.

1. The mechanism of trypsin-induced contraction in the rat myometrium was investigated using front-surface fluorimetry on fura-PE3-loaded strips. The expression of protease-activated receptors (PARs) in the rat myometrium was determined by reverse transcription polymerase chain reaction (RT - PCR). 2. In non-pregnant rats, 10 microM trypsin developed a force of up to 30.5 +/- 5.1% of that obtained during the 40 mM K(+)-depolarization-induced contraction. In pregnant rats, the maximal level of the cytosolic Ca(2+) concentration and tension obtained with 3 microM trypsin was 143.2 +/- 6.0% and 63.2 +/- 7.9%, respectively. The depletion of the extracellular Ca2+ abolished the trypsin-induced contraction. 3. Trypsin-induced contraction was abolished by the pre-treatment of a serine protease inhibitor. PAR1-activating peptide (PAR1-AP) caused a potent contraction of the myometrium, while neither PAR2-AP nor PAR4-AP induced any contraction. 4. RT - PCR analysis detected the expression of PAR1 mRNA. However, neither PAR2 nor PAR4 mRNA was detected in the rat myometrium. 5. Once the strips were stimulated with thrombin, the subsequent application of thrombin failed to induce any contraction, while trypsin induced a contraction similar to that observed without the pre-stimulation with thrombin. Once the strip was stimulated with trypsin, neither trypsin nor thrombin induced any contraction. The response to PAR1-AP remained after the pre-stimulation with thrombin and trypsin. 6. In conclusion, PAR1 was the only known receptor for trypsin expressed in the rat myometrium, but it was suggested to be cleaved and inactivated by trypsin. Trypsin was thus suggested to contract the rat myometrium via a novel type of PAR, which might be upregulated during pregnancy.

Animals↗
PubMed2003

Unproductive cleavage and the inactivation of protease-activated receptor-1 by trypsin in vascular endothelial cells.

1 Using fura-2 fluorometry of [Ca(2+)](i) in response to thrombin, trypsin and protease-activated receptor activating peptides (PAR-APs), we determined whether trypsin cleaves protease-activated receptor 1 (PAR1) and activates it in the endothelial cells of the porcine aortic valves and human umbilical vein. 2 Once stimulated with thrombin, the subsequent application of trypsin induced a [Ca(2+)](i) elevation similar to that obtained without the preceding stimulation with thrombin in the valvular endothelial cells. However, the preceding stimulation with trypsin abolished the subsequent response to thrombin, but not to bradykinin or substance P. 3 The response to PAR1-AP (SFLLRNP) was significantly (P<0.05) reduced by the preceding stimulation with thrombin and PAR1-AP in the valvular endothelial cells, while, importantly, it remained unaffected by the preceding stimulation with either trypsin or PAR2-AP (SLIGRL). The response to PAR2-AP was reduced by the preceding stimulation with trypsin and PAP2-AP. PAR1-AP attenuated the subsequent responses not only to thrombin and PAR1-AP but also to trypsin and PAR2-AP, while PAR2-AP specifically attenuated the subsequent responses to trypsin and PAR2-AP. 4 In human umbilical vein endothelial cells, a higher affinity PAR1-AP (haPAR1-AP) (Ala-pF-Arg-Cha-HArg-Tyr-NH(2)) specifically attenuated the responses to thrombin but not trypsin. On the other hand, the response to haPAR1-AP was significantly (P<0.05) attenuated by the preceding stimulation with thrombin but not trypsin. 5 In conclusion, trypsin cleaved PAR1 but did not activate it in the endothelial cells. Moreover, the trypsin-cleaved PAR1 was no longer responsive to thrombin.

Animals↗
PubMed2006

Novel carbohydrate-binding activity of pancreatic trypsins to N-linked glycans of glycoproteins.

How glycosylation affects the reactivity of proteins to trypsin is not well understood. Bovine and porcine pancreatic trypsins were discovered to bind to alpha-Man, Neu5Acalpha2,6Galbeta1,4Glc, and alpha-galactose sequences by binding studies with biotinylated sugar-polymers. Quantitative kinetic studies supported that phenylmethylsulfonyl fluoride (PMSF)-treated trypsin binds to glycolipid analogues possessing alpha-Man or alpha-NeuAc but not to those possessing beta-galactose or beta-GlcNAc residue. Enzyme-linked immunosorbent assay (ELISA) showed that trypsin binds to six kinds of biotinylated glycoproteins possessing high mannose-type and complex-type N-glycans but not to bovine submaxillary mucin, which possesses only O-glycans. Further, the binding of trypsin to glycoproteins was differentially changed by treatments with sequential exoglycosidases, endoglycosidase H, or N-glycosidase F. Quantitative kinetic studies indicated that PMSF-treated trypsin binds with bovine thyroglobulin with the affinity constant of 10(10) m(-1), which was the highest among the glycoproteins examined, and that alpha-galactosidase treatment decreased it to 10(5) m(-1). PMSF-treated trypsin bound to other glycoproteins, including ovomucoid, a trypsin inhibitor, with the affinity constants of 10(8)-10(5) mol(-1) and were markedly changed by glycosidase treatments in manners consistent with the sugar-binding specificities suggested by ELISA. Thus, the binding site for glycans was shown to be distinct from the catalytic site, allowing trypsin to function as an uncompetitive activator in the hydrolysis of a synthetic peptide substrate. Correspondingly the carbohydrate-binding activities of trypsin were unaffected by treatment with PMSF or soybean trypsin inhibitor. The results indicate the presence of an allosteric regulatory site on trypsin that sugar-specifically interacts with glycoproteins in addition to the proteolytic catalytic site.

Animals↗
PubMed2000

Do plasma and urine trypsinogen activation peptides (TAP) really increase in trypsin-taurocholate-induced pancreatitis?

Plasma and urine levels of trypsinogen activation peptides (TAP) reflect the severity of acute pancreatitis in experimental and clinical acute pancreatitis. In trypsin-taurocholate-induced pancreatitis in rats, the extrinsic bovine trypsin used for the induction of pancreatitis might influence on the TAP levels after induction of pancreatitis. The aim of the present study was to elucidate whether infused trypsin itself affects TAP levels in trypsin-taurocholate-induced pancreatitis. Rats were divided into three groups. In the pancreatitis group, acute pancreatitis was induced by a retrograde infusion of bovine trypsin and sodium taurocholate into the pancreatic duct. In the duct infusion group and peritoneal injection group, a mixture of bovine trypsin and trypsin inhibitor, ONO-3403, was infused into the pancreatic duct or the peritoneal cavity. Plasma and urine TAP concentration significantly increased in trypsin-taurocholate-induced pancreatitis but not in the duct infusion and peritoneal injection groups for 6 hours after the infusion of trypsin. Serum rat immunoreactive trypsin (IRT) and amylase significantly increased in the pancreatitis and duct infusion groups but not in the peritoneal injection group. Serum levels of bovine IRT in the pancreatitis group was significantly lower than those in duct infusion and peritoneal injection groups. In conclusion, an intraductal infusion of bovine trypsin itself into pancreatic duct does not influence the levels of plasma and urine TAP in trypsin-taurocholate-induced pancreatitis.

Acute Disease↗
PubMed1979

Isolation and characterization of trypsin-resistant O1 variants of foot-and-mouth disease virus.

Strains of foot-and-mouth disease virus of types O1 and A10 were isolated which showed no significant loss of infectivity upon trypsinization. These 'trypsin-resistant' (TR) viruses were obtained by serial passage in BHK cells of virus that was trypsin-treated before inoculation of the cells. Three O1 isolates were cloned and studied further. Cell attachment of those TR O1 variants (OTR1) was not reduced by trypsinization, unlike that of parent virus. The polypeptide compositions of TR viruses as determined by SDS-polyacrylamide gel electrophoresis were identical with those of parent virus, with the exception of OTR1 which contained an additional polypeptide approx, 3000 daltons larger than VP1. After trypsinization, which normally cleaves VP1, the polypeptide composition of the three TR viruses (including OTR1) and of parent virus did not show any significant difference. In OTR1 both the additional virus protein and VP1 were cleaved into a P18 molecule and smaller fragments. The surface location of this additional polypeptide was confirmed by iodination experiments. It was shown by immunodiffusion experiments that only OTR1 differed from the parent virus. This antigenic change was present on the trypsin-sensitive part of the virus since trypsinized TR viruses (including OTR1) were antigenically identical to trypsinized parent virus. The electrophoretic mobilities of the three OTR viruses isolated, and of parent virus, differed somewhat before trypsinization. After trypsin-treatment, the mobilities of TR viruses were all increased to the same level; however, their rate of migration was lower than that of trypsin-treated parent virus. This lower mobility of trypsin-treated OTR viruses was the only difference which could be associated with retained infectivity.

Antigens, Viral↗
PubMed2003

Plasmodium falciparum is able to invade erythrocytes through a trypsin-resistant pathway independent of glycophorin B.

Plasmodium falciparum invades erythrocytes through multiple ligand-receptor interactions, with redundancies in each pathway. One such alternate pathway is the trypsin-resistant pathway that enables P. falciparum to invade trypsin-treated erythrocytes. Previous studies have shown that this trypsin-resistant pathway is dependent on glycophorin B, as P. falciparum strains invade trypsin-digested glycophorin B-deficient erythrocytes at a highly reduced efficiency. Furthermore, in a recent study, the P. falciparum 7G8 strain did not invade glycophorin B-deficient erythrocytes, a finding that was not confirmed in the present study. To analyze the degree of dependence on glycophorin B for invasion by P. falciparum through the trypsin-resistant pathway, we have studied the invasion phenotypes of five parasite strains, 3D7, HB3, Dd2, 7G8, and Indochina I, on trypsin-treated normal and glycophorin B-deficient erythrocytes. Invasion was variably reduced in glycophorin B-deficient erythrocytes. Four strains, 3D7, HB3, Dd2, and Indochina I, invaded trypsin-treated erythrocytes, while invasion by the 7G8 strain was reduced by 90%. Among the four strains, invasion by 3D7, HB3, and Dd2 of trypsin-digested glycophorin B-deficient erythrocytes was further reduced. However, Indochina I invaded trypsin-digested glycophorin B-deficient erythrocytes at the same efficiency as its invasion of trypsin-digested normal erythrocytes. This strongly suggests that the Indochina I strain of P. falciparum is not dependent on glycophorin B to invade through a trypsin-resistant pathway as are the strains 3D7, HB3, and Dd2. Thus, P. falciparum is able to invade erythrocytes through a glycophorin B-independent, trypsin-resistant pathway.

Animals↗
PubMed1979

Serum immunoreactive trypsin concentration after a Lundh meal. Its value in the diagnosis of pancreatic disease.

The changes in serum trypsin concentration have been measured in 47 subjects for up to 2 hours after a Lundh meal. In 18 healthy controls, mean fasting trypsin concentration was 285 +/- 125 ng/ml (mean +/- 2 SD). The maximum increase after the Lundh meal (the trypsin response ratio) was 6.7 +/- 7.5%. Six patients with chronic renal failure had elevated fasting serum trypsin concentrations (range 460-1100 ng/ml) but trypsin response ratios fell within the control range. Of five patients with relapsing pancreatitis, two had raised and three normal or low fasting trypsins. After stimulation two had elevated trypsin response ratios; one of the two had evidence of main duct obstruction. Eleven out of 12 patients with chronic pancreatitis (with or without insufficiency) had low fasting trypsin concentrations (range 0-120 ng/ml) Seven of the 12 also had raised trypsin response ratios. In six patients with cancer of the pancreas, fasting trypsin was low in three, normal in two, and raised in one. Both patients with a normal fasting level had a raised trypsin response ratio. The combination of a single estimation of fasting serum trypsin concentration followed by serial measurements after a Lundh meal provides a useful screening test for chronic pancreatic disease.

Chronic Disease↗
PubMed1991

Liver uptake of trypsin-inhibitor complexes; differences between suckling and adult rats.

Previous studies have demonstrated increased intestinal trypsin uptake in newborn rats compared to adults. The mechanisms that protect tissues against proteolytic damage by trypsin include trypsin-inhibitor binding and subsequent liver uptake. In order to examine these mechanisms, bovine trypsin (1.25 mg/100 g body weight) plus trace 125I-trypsin were injected into the portal vein of 2-week-old and adult rats. Liver, kidney and plasma 125I activity were assessed at 1, 5 or 15 min following infusion and the different trypsin inhibitor fractions were separated and examined for 125I activity. Newborn rats had significantly increased plasma levels of 125I-trypsin and significantly decreased liver 125I levels 1 min after infusion compared to adults. In addition, the slow decline in liver 125I activity seen in the adult rats did not occur in the newborns. The pattern of trypsin-inhibitor binding was not significantly different at 1, 5 and 15 min and there were no differences at these time intervals between newborns and adults. We suggest that the newborn liver is less effective in clearing infused trypsin leading to increased plasma levels, and this increased plasma trypsin concentration subsequently leads to increased liver levels of 125I-trypsin. The increased trypsin levels in the liver may predispose newborns to protease-induced liver damage.

Acute-Phase Proteins↗
PubMed1986

Leukocytes are required for the trypsin-induced increase in lung vascular permeability.

The authors examined the role of leukocytes in mediating the increase in lung vascular permeability induced by trypsin infusion in the sheep lung lymph preparation. One group of sheep was challenged with an intravenous infusion of trypsin (4.5 mg/kg/hr). A second group was depleted of 80% of circulating granulocytes and of 48% of circulating lymphocytes by repeated injections of hydroxyurea several days prior to the trypsin infusion. Pulmonary lymph flow and transvascular protein clearance increased twofold without changes in pulmonary vascular pressures in the control group, suggesting that trypsin resulted in an increase in pulmonary vascular permeability. The hydroxyurea-induced leukopenia prevented the increases in pulmonary lymph flow and protein clearance after the trypsin infusion, indicating that leukocytes are required for increase in lung vascular permeability. Because neutrophil activation may mediate the trypsin-induced increase in lung vascular permeability, we assessed the effect of trypsin on superoxide anion (O2-) generation by isolated neutrophils. Trypsin (0.09 mg/ml) added to isolated sheep neutrophils did not increase O2- generation more than neutrophils in buffer. The supernatant obtained after incubation of trypsin with citrated whole blood increased O2- generation from isolated neutrophils, this response was greater than with trypsin alone. Therefore, neutrophil activation occurs as a result of the action of trypsin on whole blood. Neutrophil activation may contribute to the leukocyte-dependent increase in lung vascular permeability after trypsin.

Animals↗
PubMed1982

[Evaluation of measuring serum trypsin by radioimmunoassay and clinical application for the study on the exocrine function in diabetics (author's transl)].

Measurement of serum concentration of trypsin by RIA-Gnost Trypsin kit (Hoechst-Japan) was evaluated. The clinical usefulness of measuring serum trypsin level in diabetic patients was assessed. The measurement of trypsin using the radioimmunoassay (RIA) kit revealed good precision and reproducibility with intraassay error ranging from 3.6 to 5.5% in C.V. corresponding to mean trypsin concentration of 236.5-838.7 ng/ml and interassay error ranging from 8.1 to 11.1%. Tests for recovery and dilution were satisfactory for clinical use. Clinical materials included 35 normal subjects, 88 diabetics, 22 patients with liver diseases, 3 with acute pancreatitis, 7 with chronic pancreatitis and 3 with chronic renal failure. Serum trypsin concentration in normal controls was 157.6 +/- 59.9 ng/ml (m + 1 S.D.). Diabetic patients treated with diet therapy alone revealed serum trypsin level of 203.6 +/- 74.8 ng/ml (n = 50). In diabetics treated with sulfonil urea serum trypsin was 171.3 +/- 83.0 ng/ml (n = 25). In patients receiving insulin serum trypsin level was 90.5 +/- 49.0 ng/ml (n = 13). In patients with liver diseases, acute pancreatitis, chronic pancreatitis and chronic renal failure serum trypsin concentration were 236.9 +/- 88.0, 520.1 +/- 80.0, 113.0 +/- 75.6, and 2557 +/- 2771 respectively. Our results may indicate impaired pancreatic exocrine function in patients with severe diabetes mellitus. Increased serum trypsin level in diabetics treated with diet therapy may be due to stimulated excretion of trypsin resulted from restricted food intake. However, further study in larger number of patients is needed.

Diabetes Mellitus↗
PubMed1994

The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line.

The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel.

ATP-Binding Cassette Transporters↗
PubMed1976

Disulfide bond-modified trypsinogen. Role of disulfide 179-203 on the specificity characteristics of bovine trypsin toward synthetic substrates.

Disulfide 179-203 of trypsinogen was cleaved and the free sulfhydryls were modified by S-carboxymethylation, S-carboxyamidomethylation, or by S-aminoethylation. The enzymatic properties of the activated, modified zymogens toward specific and nonspecific trypsin substrates were studies. The three S-alkylated trypsins hydrolyzed N-benzoylarginine ethyl ester and N-tosyl-lysine methyl ester with Kcat values similar to those of trypsin but with Km values that were increased by 2 to 3 orders of magnitude. The binding constant of the competitive inhibitor benzamidine to the S-alkylated trypsins was increased by 2 orders of magnitude by the modifications. The association constant of soybean trypsin inhibitor with S-carboxyamidomethyl trypsin was several orders of magnitude less than normal. Hydrolysis of benzoyl arginine amide with S-carboxymethyl trypsin was not detected, but this was ascribed to poor binding since a KI of 0.3 M was estimated from competitive inhibition studies. The altered kinetics did not depend on the type of chemical group used for the S-alkylated derivative, nor were the kinetics of any derivative significantly influenced by changes of pH or ionic strength. Nonspecific substrates, such as acetylglycine p-nitrophenyl ester and p-nitrophenylacetate, were hydrolyzed at equal rates by both trypsin and disulfide-modified trypsins, and both substrates had identical kcat/Km ratios with unmodified trypsins. For S-alkylated trypsins, kcat/km with lysine and arginine substrates were lower than normal and were the same order of magnitude as the values found for nonspecific substrates, suggesting normal catalytic behavior but a loss of specificity in binding substrates. The kinetic evidence suggested that the role of disulfide 179-203 in the mechanism of action of trypsin is to maintain the geometry of the specificity pocket by keeping appropriate residues of the pocket in a rigid framework.

Animals↗
PubMed1988

Purification and characterization of two trypsin-like enzymes from the digestive tract of anchovy Engraulis encrasicholus.

1. Two trypsin-like enzymes, designated Trypsin A and B, were purified from the pyloric caeca and intestine of anchovy by (NH4)2SO4 fractionation, affinity chromatography (Benzamidine-Sepharose-6B) and ion exchange chromatography (DEAE-Sepharose). 2. Both trypsins catalyzed the hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), p-tosyl-L-arginine methyl ester (TAME), casein and myofibrillar protein and they were inhibited by several well established trypsin-inhibitors. 3. The enzymes had mol. wts of 27,000 (Trypsin A) and 28,000 (Trypsin B). Their isoelectric points were about 4.9 (Trypsin A) and 4.6 (Trypsin B) and they had similar amino acid composition. 4. The enzymes had a pH optimum of 8-9 for the hydrolysis of BAPNA and of 9.5 for the digestion of casein and myofibrillar protein. Their activity and stability were affected by calcium ions. 5. Trypsins A and B resemble other fish trypsins in their mol. wt, pI, kinetic properties and the instability at low pH and they are similar to bovine trypsin in their dependence of Ca2+ for activity and stability.

Amino Acids↗
PubMed1987

Interaction of silkworm larval hemolymph antitrypsin and bovine trypsin.

Antitrypsin isolated from silkworm larval hemolymph can inhibit bovine trypsin. The apparent molar ratio of silkworm antitrypsin (sw-AT) to trypsin at extrapolated null trypsin activity was determined to be 1.3 by titration of trypsin with sw-AT. The undissociability of the complex between sw-AT and trypsin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was confirmed by immunoblotting and fluorescence labeling techniques. Chemical analysis of the complex elucidated that it contained equimolar sw-AT and trypsin. Densitometric analysis of electrophoretic patterns obtained during the titration of trypsin by sw-AT suggested the presence of a suicide product formed from sw-AT. This was the reason why excess sw-AT was needed for complete inhibition of trypsin. In the complex, the sw-AT molecule was cut at one site but the fragments produced were still joined together. Trypsin in the complex was released by treatment at pH 10.0, and it was deduced that the complex formation involved acyl-bond formation between sw-AT and trypsin. The sw-AT component obtained from the alkali-treated complex possessed two kinds of NH2-terminal amino acid sequences. Non-covalent forces may bind the two fragments of sw-AT, which could be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequence analysis of the large fragment gave a sequence identical with that of intact sw-AT. This indicated that the reactive site of sw-AT with trypsin was located at the COOH-terminal region of the molecule. These characteristics resemble those of inhibitors belonging to the serpin family.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of trypsin from the poikilotherm Gadus morhua.

A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularly with dogfish and some mammalian trypsins. The apparent Km values determined at 25 degrees C for the predominant form of Atlantic cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 microM and 77 microM respectively, which are notably lower values than those determined for bovine trypsin (46 microM and 650 microM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.

Amidohydrolases↗
PubMed1976

[The inter-alpha-trypsin inhibitor as precursor of the acid-stable proteinase inhibitors in human serum and urine].

A small amount of antitryptic activity is detectable in the supernatant of deproteinized human serum. Preincubation of serum with trypsin causes an increase in acid-stable antitryptic activity. This rise in activity depends on the inter alpha-trypsin inhibitor concentration. The native inhibitor present in normal sera, and in higher concentrations in sera of patients with nephropathies, and the trypsin-liberated inhibitor show immunological cross reaction with antibodies to the serum inter-alpha-trypsin inhibitor. The two inhibitors differ in molecular weight and electrophoretic mobility. The physiological inhibitor (I-34), with a molecular weight of 34 000 and a high carbohydrate content, can be transformed by trypsin into an inhibitor (I-17) with a molecular weight of 17 000. This inhibitor is identical with the inhibitors liberated by trypsin from serum or from purified inter-alpha-trypsin inhibitor. The acid-stable inhibitor from urine is identical with the physiological serum inhibitor. Analogously, this inhibitor is transformed by trypsin into the inhibitor with a molecular weight of 17 000. We conclude that the inter-alpha-trypsin inhibitor is the precursor of both the physiological and the trypsin-liberated inhibitor. By a mechanism as yet unknown, but most likely a limited proteolysis, the secreted inhibitor is liberated from the high molecular weight precursor. In contrast to the monospecific trypsin-inhibiting precursor, the physiological and artificially liberated inhibitors are trypsin/chymotrypsin/plasmin inhibitors.

Humans↗
PubMed1987

Three high molecular weight protease inhibitors of rat plasma. Reactions with trypsin.

We have compared the reactions of trypsin with human alpha 2-macroglobulin (alpha 2M), and three rat plasma protease inhibitors, alpha 1-macroglobulin (alpha 1M), alpha 1-inhibitor III (alpha 1I3), and alpha 2M. All four of these proteins appear to contain reactive thiol esters. The electrophoretic mobility in agarose gels of human and rat alpha 2M is increased by 1 mol of trypsin, while the mobility of alpha 1M and alpha 1I3 is decreased. Treatment with methylamine causes similar mobility changes, except in the case of rat alpha 2M. Titration of human and rat macroglobulins by repeated small additions of trypsin and by assay of liberated SH groups or enhanced ligand fluorescence revealed a stoichiometry of about 1 mol of trypsin/mol of inhibitor. In contrast, addition of macroglobulin to a fixed amount of trypsin and detection of residual amidase or protease activity revealed a stoichiometry of about 2 mol of trypsin for 1 mol of human alpha 2M, about 1.4 mol for rat alpha 1M, and about 1 mol for rat alpha 2M. One mol of trypsin reacted with 2 or more mol of alpha 1I3 by the criteria of SH groups liberated or protease inhibition. Methylamine-treated rat alpha 2M binds a significant amount of trypsin releasing about 2 mol of SH. Radioactive beta-trypsin was covalently bound to subunits of the purified plasma inhibitors. The Mr of the labeled products with rat and human alpha 2M had molecular weights which suggested trypsin was bound to intact as well as cleaved subunit chains and also to multiple chains via cross-linking. Rat alpha 1M also produced a product which may be an intact subunit alpha chain plus trypsin. Greater than 80% of the trypsin was bound covalently to these inhibitors at low molar ratios.

Acute-Phase Proteins↗