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At least 37 records · Page 2Linked to original sources
PubMed1985

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, X. The amino-acid sequences of the trypsin-released inhibitors from horse and pig inter-alpha-trypsin inhibitors.

The amino-acid sequences of the acid-resistant inhibitors released from horse and pig inter-alpha-trypsin inhibitor (ITI) by tryptic proteolysis were determined. They are composed of two covalently linked Kunitz-type domains. In both cases the reactive site of their C-terminal antitryptic domains is occupied by arginine as in the homologous human and bovine inhibitors. The reactive site of their N-terminal domain exhibits only a weak interaction with polymorphonuclear granulocytic elastase and is occupied by leucine as in the strong elastase inhibitor released from bovine ITI. The differences between inhibitory activities of the ITI-derived inhibitors from horse, pig, and cattle are discussed on the basis of sequence differences in position P'2.

Alpha-Globulins↗
PubMed1996

Protective effects of human urinary trypsin inhibitor against trypsin-induced relaxation in rat aorta.

OBJECTIVES: To investigate the vasodilatory responses induced by trypsin, and to determine the antitrypsin effects of human urinary trypsin inhibitor on blood vessels. DESIGN: Prospective, randomized, controlled study, with repeated measurements. SETTING: University research laboratory. INTERVENTIONS: The isometric tension of the aortic rings isolated from male Wistar rats was recorded. The vasodilatory responses to varying amounts of trypsin (0.01 to 10 U/mL) were examined under pretreatment with human urinary trypsin inhibitor (1 to 10 U/mL), chicken-egg-white trypsin inhibitor (0.1 to 10 mg/mL), N infinity-nitro-L-arginine (3 x 10(-5) M), and denudation of the endothelium. In addition, with the scanning electron microscopy, the endothelium of aorta was examined under treatment with trypsin alone (10 U/mL), and with trypsin plus human urinary trypsin inhibitor (10 U/mL each).4 MEASUREMENTS AND MAIN RESULTS: The addition of trypsin produced dose-dependent relaxation in aortic rings with an intact endothelium, which was abolished by denudation and treatment with N infinity-Nitro-L-arginine. Pretreatment with human urinary trypsin inhibitor and with chicken-egg-white trypsin inhibitor caused dose-dependent inhibition against trypsin-induced vasorelaxation. On the repeated trials of the response to trypsin, the responses gradually developed tachyphylaxis in control aortic rings, whereas no tachyphylaxis developed in rings pretreated with human urinary trypsin inhibitor (10 U/mL). Scanning electron microscopy demonstrated endothelial disruption in aorta exposed to trypsin alone, whereas the endothelium was intact in strips treated with trypsin plus human urinary trypsin inhibitor. CONCLUSIONS: The data indicate that relaxation induced by trypsin is attributable to nitric oxide released from the endothelium, and that human urinary trypsin inhibitor protects vessels against trypsin-induced endothelial injury. It appears that the clinical antishock effect of human urinary trypsin inhibitor is ascribable in part to its antitrypsin activity on the endothelium of vascular smooth muscle.

Animals↗
PubMed2006

Reevaluation of the role of early trypsin activity in the transcriptional activation of the late trypsin gene in the mosquito Aedes aegypti.

In the female mosquito Aedes aegypti, trypsin expression is largely biphasic. Early trypsin synthesis, which is regulated at the translational level relative to feeding, peaks in the first few hours post-blood meal. Late trypsin expression is regulated at the transcriptional level, and peaks 18-24h post-blood meal. It was proposed that early trypsin activity released unknown factors during digestion of a meal that caused activation of transcription of the late trypsin gene. This connection between early trypsin activity and late trypsin expression was dependent on the fact that feeding a single trypsin inhibitor, soybean trypsin inhibitor (STI), which blocked early trypsin activity, also blocked late trypsin expression. We show in this study that feeding different trypsin inhibitors which effectively blocked early trypsin activity did not result in reduced late trypsin expression. We also found that a different lot of STI failed to cause inhibition of late trypsin transcription, although it was effective in inhibiting early trypsin activity. In addition, using RNAi methodology to reduce the level of early trypsin expression had no effect on the level of late trypsin expression. We conclude that early trypsin activity is not necessary for the transcriptional activation of late trypsin and that the previous results were due to the effect of a cytotoxic agent present in some, but not all preparations of STI.

Aedes↗
PubMed1975

Preferred binding of bovine and porcine trypsins at two different sites on chicken ovoinhibitor. Reduced dissociation of mixed trypsin complexes.

Dissociation of mixed trypsin (bovine plus porcine trypsin) complexes with chicken ovoinhibitor was used to investigate the nonequivalence of the two binding sites for trypsin on the inhibitor. Previous work has shown that 1 mol of trypsin dissociates much more rapidly than the 2nd from unmixed trypsin complexes, those containing 2 mol of one kind of trypsin, bovine or porcine, per mol of inhibitor. However, only approximately 0.5 to 0.6 mol of trypsin dissociated in the rapid step from the mixed trypsin complexes, those containing 1 mol each of bovine and porcine trypsin. Rates of the slow dissociation steps for the two types of complexes did not differ appreciably from each other. A general dissociation scheme is proposed, in which each of the 2:1 complexes can lose a trypsin molecule from either in two parrallel first order reactions, producing two different 1:1 complexes, which subsequently dissociate to yield free ovoinhibitor and a second trypsin molecule. In this scheme, both the earlier results with unmixed trypsin complexes and the preponderance (approximately 3:1) of slow dissociation from the mixed trypsin complexes can be rationalized if bovine trypsin is retained preferentially at one of the two trypsin binding sites on chicken ovoinhibitor, and porcine trypsin at the other. That is, one site allows rapid dissociation of porcine trypsin and slow dissociation of bovine trypsin, whereas the other allows rapid dissociation of bovine trypsin and slow dissociation of porcine trypsin.

Animals↗
PubMed1983

Enzymatic properties of alpha 2-macroglobulin-proteinase complexes. Apparent discrimination between covalently and noncovalently bound trypsin by reaction with soybean trypsin inhibitor.

The binding of trypsin to alpha 2-macroglobulin, the appearance of free beta-cysteinyl thiol groups of the formed complexes, the steady-state kinetics of their enzymic hydrolysis of carbobenzoxy-L-valyl-glycyl-L-arginyl-4-nitroanilide and finally their reactions with soybean trypsin inhibitor leading to the formation of ternary alpha 2-macroglobulin-trypsin-soybean trypsin inhibitor complexes were investigated. Each alpha 2-macroglobulin molecule binds two trypsin tightly; the dissociation constants were found to be unmeasureably small, but the extent of formation of 1:1 and 1:2 complexes at different molar ratios of alpha 2-macroglobulin to trypsin as determined from the appearance of thiol groups clearly indicated that binding of trypsin to alpha 2-macroglobulin shows negative cooperativity. Binding of the first trypsin makes the access of the second less easy. The kinetic results showed a decrease of the kc/Km value of hydrolysis of the tripeptide substrate by approx. 4-fold compared to that of free trypsin for each alpha 2-macroglobulin-bound trypsin. Here no differences were seen between the bound trypsins. The analysis of the reactions between the alpha 2-macroglobulin-trypsin complexes and soybean trypsin inhibitor shows that ternary complexes do form, although slowly, and that two processes occur, not only when 1:2 complexes but also when 1:1 complexes react with soybean trypsin inhibitor. Soybean trypsin inhibitor apparently discriminates between two distinct binding modes of trypsin to alpha 2-macroglobulin, the covalently and the noncovalently alpha 2-macroglobulin-bound trypsins.

Animals↗
PubMed1982

Interaction of urinary trypsin inhibitor, UTI68, with bovine trypsin.

One molecule of UTI68, a trypsin inhibitor purified from urine of healthy men, inhibited four molecules of bovine trypsin. This finding suggests the formation of various complexes of UTI68 with 1 to 4 molecules of trypsin. However, SDS polyacrylamide gel electrophoresis of the reaction products of UTI68 with trypsin showed that, at molecular ratios of UTI68 to trypsin of 1:1 to 1:3, UTI68 was rapidly cleaved by trypsin to form two proteins, Protein I and Protein II, whose molecular weights were estimated as 49,000 and 25,000, respectively, while at a ratio of 1:4, UTI68 was converted to Protein III with a molecular weight of about 30,000 and a smaller protein(s) than trypsin. These results were supported by gel filtration of the reaction products of UTI68 with trypsin on a Sephadex G-100 column at pH 3.0. Protein I and Protein II were separated, and Protein I was named UTI49. Protein II was separated from trypsin on a QAE-Sephadex column, and it had no inhibitory activity. Since one molecule of UTI49 inhibited about three molecules of trypsin, its interaction with trypsin was examined. On addition of one and two molecules of trypsin to one molecule of UTI49 at pH 8.0 complexes were formed consisting of one and two molecules of trypsin, respectively, with one molecule of UTI49, and both complexes were dissociated to their components at pH 3.0. Addition of three molecules of trypsin brought about further fragmentation of UTI49, and the split products formed a complex(es) with trypsin at pH 8.0, which dissociated at pH 3.0.

Animals↗
PubMed1996

Dietary modulation of the mRNA stability of trypsin isozymes and the two forms of secretory trypsin inhibitor in the rat pancreas.

The stability of the mRNAs encoding pancreatic trypsin isozymes, namely the cationic form and the two anionic forms I and II, as well as that of the secretory trypsin inhibitors I and II, were studied in rats fed on either a high-protein diet, or a protein-free diet compared with a standard diet for a 10-day period. Either immediately or 3 h and 6 h after injecting the transcription inhibitor, actinomycin D, the mRNA levels were quantified by performing dot-blot hybridization with specific oligonucleotide probes. Under high-protein dietary conditions, the stability of the mRNAs coding for anionic trypsin II and cationic trypsin showed no change, whereas that of anionic trypsin I and the two forms of secretory trypsin inhibitor were affected. The mRNA half-life of anionic trypsin I and trypsin inhibitor II increased, in sharp contrast with that of trypsin inhibitor I, which decreased. When rats were fed on a protein-free diet, the stabilities of both anionic trypsin forms and trypsin inhibitor I increased, whereas that of trypsin inhibitor II decreased and that of cationic trypsin remained unchanged. The present results show the existence of differences in the mechanisms whereby gene expression of trypsin isozymes and secretory trypsin inhibitors is regulated, although they are synthesized in parallel in the pancreatic acinar cell and stored in zymogen granules before being secreted into the intestinal lumen.

Animals↗
PubMed1987

Effectiveness of Sepharose-bound trypsin versus liquid-phase trypsin plus benzamidine for activation of inactive renin in human plasma.

We compared the effectiveness of two techniques involving the use of the enzyme trypsin to activate inactive renin in human plasma. Both these methods were developed to optimize activation with trypsin by preventing the possible destruction of activated renin by trypsin itself. In one method, an antitryptic agent such as benzamidine is added to plasma, concomitantly with trypsin (liquid phase). In the other a low concentration of Sepharose-bound (immobilized) trypsin is used. In six plasma samples we have found that trypsin (1.5 mg/ml) activation, with or without benzamidine (0.8 mg/ml), yielded similar values of activated renin (11.0 +/- 2.7 vs. 11.3 +/- 2.3 ng/ml/hr). However, the addition of immobilized trypsin to pool plasma pretreated with trypsin plus benzamidine caused a further increase in plasma renin activity (PRA); in contrast, the addition of trypsin and benzamidine to pool plasma pretreated with immobilized trypsin caused a decrease in PRA. In 17 plasma samples from patients with essential hypertension we found that the inactive renin values were always higher after treatment with immobilized trypsin than with trypsin plus benzamidine (9.0 +/- 0.7 vs. 6.1 +/- 0.5 ng/ml/hr, P less than 0.01); moreover, there was a positive correlation between the differences in the values of inactive renin measured with the two methods and the values obtained with immobilized trypsin (r = 0.64, P less than 0.01). Therefore, the activation with immobilized trypsin is more effective than that with liquid-phase trypsin, alone or in combination with benzamidine, in converting inactive renin in human plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidines↗
PubMed1991

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).

Animals↗
PubMed1999

High-resolution structures of three new trypsin-squash-inhibitor complexes: a detailed comparison with other trypsins and their complexes.

An anionic trypsin from Atlantic salmon and bovine trypsin have been complexed with the squash-seed inhibitors, CMTI-I (Cucurbita maxima trypsin inhibitor I, P1 Arg) and CPTI-II (Cucurbita pepo trypsin inhibitor II, P1 Lys). The crystal structures of three such complexes have been determined to 1.5-1.8 A resolution and refined to crystallographic R factors ranging from 17.6 to 19.3%. The two anionic salmon-trypsin complexes (ST-CPTI and ST-CMTI) and the bovine-trypsin complex (BT-CPTI) have been compared to other trypsin-inhibitor complexes by means of general structure and primary and secondary binding features. In all three new structures, the primary binding residue of the inhibitor binds to trypsin in the classical manner, but with small differences in the primary and secondary binding patterns. Lysine in CPTI-II binds deeper in the specificity pocket of bovine trypsin than lysine in other known lysine-bovine-trypsin complexes, and anionic salmon trypsin lacks some of the secondary binding interactions found in the complexes formed between squash inhibitors and bovine trypsin. The ST-CMTI complex was formed from the reactive-site-cleaved form of the inhibitor. However, well defined electron density was observed for the P1-P1' peptide bond, together with a hydrogen-bonding pattern virtually identical to those of all serine-protease-protein-inhibitor complexes, indicating a resynthesis of the scissile bond.

Amino Acid Sequence↗
PubMed1989

Trypsin uptake and trypsin-inhibitor complexes. Differences between suckling and adult rats.

Previous studies have suggested that both newborn animals and humans absorb intact proteases from the intestine in greater amounts than do adults. Absorbed proteases are rapidly complexed in plasma by several inhibitors which inactivate free proteases. In order to confirm increased intestinal uptake in newborns, we evaluated the plasma trypsin activity in both 2-week-old and adult rats following a trypsin feed. In addition, we studied the interaction between absorbed trypsin and rat trypsin inhibitors (alpha-macroglobulin, alpha 1-inhibitor 3, and alpha 1-protease inhibitor) by determining the concentration of alpha-macroglobulin complexes both in vivo and in vitro following trypsin feeding and by evaluating the amount of trypsin activity complexed with the different inhibitors. In vivo experiments demonstrated that trypsin uptake was significantly increased in newborns compared to adult rats and that 50-70% of plasma trypsin activity was associated with alpha 1-inhibitor 3. Increased uptake was not accompanied by increased alpha-macroglobulin complexes. In vitro trypsin incubation did not lead to increased alpha-macroglobulin complexes until the other inhibitors were removed. These findings suggest that newborns have increased trypsin uptake, and the trypsin initially binds to alpha 1-inhibitor 3 before being transferred to alpha-macroglobulin for clearance. Further studies are needed in order to understand the interaction between intestinal uptake and plasma inhibition of absorbed proteases.

Aging↗
PubMed2003

Co-expression of trypsin and tumour-associated trypsin inhibitor (TATI) in colorectal adenocarcinomas.

Trypsin and its specific inhibitor, TATI (tumour-associated trypsin inhibitor), are expressed in normal human pancreas and in a variety of tumours. The aim of the present study was to assess the parallel expression of trypsin and TATI in colorectal cancer, in comparison with their expression in normal epithelial tissue, since proteases and their inhibitors are thought to be co-expressed in malignant neoplasms. We also assessed the possible significance of their expression as a means of differentiation between normal and malignant tissue. We examined qualitatively and semi-quantitatively the immunohistochemical expression of trypsin and TATI on paraffin-embedded serial tissue sections from 91 colorectal adenocarcinomas. The reverse-transcriptase-polymerase-chain reaction (RT-PCR) was also performed on fresh malignant tissue from 55 of the above adenocarcinomas. Normal and non-malignant tissues adjacent to the tumours were also evaluated. Cytoplasmic expression of trypsin (more than 25% of the cancer cells positive) was found in 67 (73.6%) adenocarcinomas, whereas TATI was expressed in the cytoplasm of 59 (64.8%) cases studied. Statistical analysis using Spearman's test has demonstrated a significant correlation between trypsin and TATI immunohistochemical expression (p<0.01). RT-PCR showed co-expression of trypsin and TATI mRNA in all carcinomas studied. Distinct patterns of trypsin and TATI immunohistochemical expression were observed in adjacent, non-malignant tissues, where both trypsin and TATI mRNA were also detected. Normal tissues were negative by immunohistochemistry. Our results indicate co-expression of trypsin and TATI in colorectal tumours both at the mRNA and protein level. We conclude that in colorectal neoplasms, high levels of trypsin and TATI may be important for malignant tumour formation and/or metastatic process.

Adenocarcinoma↗
PubMed1996

Molecular sequencing and modeling of Neobellieria bullata trypsin. Evidence for translational control by Neobellieria trypsin-modulating oostatic factor.

Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reversed transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3' end of the gene (300 bp) was amplified by means of the rapid-amplification-of-cDNA-ends method, cloned and sequenced. The deduced protein sequence of 254 amino acids exhibited 46% identity to Drosophila trypsin and 32% identity to Anophiline trypsin and Aedes trypsin. Three-dimensional models of Neobellieria trypsin and Drosophilia trypsin were built and compared. Both models contain two domains of beta-barrel sheets as was shown by means of X-ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Ser182 whereas Asp176 sits as the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugar-fed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin-modulating oostatic factor (TMOF) at 10(-9) M prevented the disappearance and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.

Amino Acid Sequence↗
PubMed2001

Transcriptional induction of diverse midgut trypsins in larval Agrotis ipsilon and Helicoverpa zea feeding on the soybean trypsin inhibitor.

Midgut trypsins insensitive to inhibition by the soybean trypsin inhibitor (STI) were found to be transcriptionally regulated in A. ipsilon and H. zea larvae feeding on STI, as demonstrated by injections with actinomycin, a transcriptional inhibitor, which abolished the production of these STI-insensitive trypsins. The induced, STI-insensitive trypsins differed from the constitutive, STI-sensitive trypsins in their susceptibility to inhibitors based on sizes, suggesting that the induced enzymes limited access to their active site by blocking bulky inhibitors. Twenty midgut cDNA fragments(1) were amplified using trypsin-specific PCR primers and at least twelve were shown to encode structurally diverse trypsins. High sequence diversity was observed for both the enzymes encoded by STI-induced mRNAs and those from larvae that had not been exposed to STI. Northern blots showed that midgut mRNAs hybridizing to various trypsin cDNA probes were either transcribed de novo or up-regulated following ingestion of STI. Southern hybridizations indicated the presence of multiple trypsin gene families in the insect genomes. The complete sequence of a trypsin gene(1) from A. ipsilon (AiT9) revealed the presence of three introns. Comparison of 5' upstream sequences(1) from AiT9 and AiT6 genes from A. ipsilon revealed putative TATA box and disparate regulatory motifs, within 500 bp of each translational start site.

Amino Acid Sequence↗