Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Virions of two strains of tobacco mosaic virus (U1 and Cc) have associated with them a small amount of a minor protein called H protein (A. Asselin and M. Zaitlin, 1978, Virology 91, 173-181), now known to be related to the viral coat protein (C.W. Collmer, V.M. Vogt, and M. Zaitlin, 1983, Virology 126, 429-448.). In the present study, a quantification technique involving disruption of virions followed by direct analysis of the component parts on SDS polyacrylamide gels was used to confirm an average of one molecule of H protein per virion for U1 TMV. H protein was separated from coat protein and purified by electrofocusing in a flatbed of granulated gel under stringent dissociating conditions. When assayed in the presence of urea, H protein has a pI of approximately 5.4, coat protein has a pI of approximately 4.9. Proteinase K-treated TMV RNA and H-protein-free TMV coat protein were reconstituted in vitro with or without H protein and the resulting virions were analyzed. A small amount of H protein reassociated with virions reconstituted in vitro (less than 10% of the amount found in native virions) and became resistant to degradation by trypsin, but such virions were no different from virions reconstituted without H protein in terms of yield of reconstituted particles or infectivity. In mixed reconstitution experiments with RNA and coat protein from strains U1 and Cc in all four possible combinations and with U1 H protein, the H protein always associated with the U1 coat protein. This demonstrated U1-H protein affinity for a specific coat protein rather than a specific RNA. It is unlikely that H protein functions in the early stages of viral infection, although the possibility of its having some other role in the life cycle of TMV remains.
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PMID:The H protein isolated from tobacco mosaic virus reassociates with virions reconstituted in vitro. 685 92

The substrate specificity of proteinase B (EC 3.4.22.9) from Baker's yeast was studied. Experiments with unblocked synthetic peptides indicated that the enzyme has no aminopeptidase activity. The proteinase cleaves trypsin substrates like Bz-Arg-OEt, Bz-Arg-pNA and Bz-Ile-Glu-Gly-Arg-pNA and chymotrypsin substrates like Ac-Tyr-OEt and Bz-Tyr-pNA. The Km value for Ac-Tyr-OEt is similar to that of chymotrypsin A, but the catalytic activity per mol proteinase B amounts to only 1/20 that of chymotrypsin A. Km and kcat for Bz-Arg-OEt are 1/50 and 1/7 as high as the corresponding values determined for trypsin. Proteinase B cleaved the oxidized insulin B chain with an initial rapid cleavage step at Leu(15)-Tyr(16) and Phe(24)-Phe(25). Slower hydrolysis was observed at Gln(4)-His(5), Leu(11)-Val(12) Tyr(16)-Leu(17), Leu(17)-Val(18), Arg(22)-Gly(23) and Phe(25)-Tyr(26). These results suggest that the specificity of proteinase B is comparable to the specificity of porcine chymotrypsin C as well as of trypsin. When the hexapeptide Leu-Trp-Met-Arg-Phe-Ala was used as a substrate for proteinase B, the enzyme preferentially attacked at Arg-Phe and more slowly at Trp-Met.
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PMID:The substrate specificity of proteinase B from baker's yeast. 702 21

Proteinase inhibitors adsorbed from human serum on DEAE Sephadex A 25 0.25 or 0.3 mol/l NaCl were purified by affinity chromatography on a trypsin-Sepharose 4B column, by gel filtration, and by DEAE cellulose chromatography. Small amounts of TCI-I (desorbed from the ion-exchange between 0.25-0.3 mol/l NaCl), and TCI-II (desorbed at NaCl concentration above 0.3 mole/l) with high specific activity were obtained. The low recovery of inhibitory activity (below 9% was due to a molecular transformation of these inhibitors after contact with the immobilized trypsin. The low-molecular weight derivatives were formed that lost their ability to adsorbe on ion-exchanger at 0.25 or 0.3 mole/l salt concentration.
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PMID:Further studies on proteinase inhibitors separated from human serum by deae sephadex chromatography. 744 41

We have examined the effects of seven proteases on human placental tissue factor in Triton X-100, focusing on extracellular and cytoplasmic domains recognized by monoclonal antibodies HTF1, C28 1.1, and C28 2.1. Plasmin produced peptides recognized on Western blots by C281.1 but not HTF1. None of the other proteases destroyed the extracellular epitope without also removing the cytoplasmic epitope, and both trypsin and chymotrypsin removed the cytoplasmic epitope with little effect on the extracellular domain. Proteinase K destroyed both epitopes, as did neutrophil elastase when used at a relatively high concentration. When digests were sampled over time and reconstituted with lipids for determination of tissue factor activity, only proteinase K consistently produced a loss in tissue factor activity at four hours. After 24 hr, other enzymes also decreased the recovered activity, with the order of effectiveness elastase > trypsin > chymotrypsin.
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PMID:Human placental tissue factor: protease susceptibility of extracellular and cytoplasmic domains. 750 71

We previously produced evidence that the human mammary-carcinoma cell line 8701-BC expresses several metalloproteinases (MMP-1, -2, -9, and -10) and their tissue inhibitors). In order to obtain a better understanding of the environmental control over gelatinolytic activities, we have tested the enzyme production of 8701-BC cells, at time intervals after plating on different collagen substrates, i.e., types I, III, IV, V and OF/LB, used as films in culture dishes. Proteinase activities, released in the conditioned culture media, were tested by zymography on SDS-PAGE, and by quantificative analyses, using 14C carboxymethylated transferrin as substrate in a liquid incubation medium. Enzymatic activities varied with time and were inversely related to cell densities, with minimum values at cell confluence. The enzymatic activity was positively supported by collagen substrates, with a maximal increase in activity when OF/LB collagen was used. In addition to the known MMPs, we found a proteinase with an M(r) of about 20 kDa, which displayed higher activity at 48 hr after cell plating and gradually decreased with cell increment. In contrast to the other MMPs, this proteinase is inhibited by soybean trypsin inhibitor, but it does not display a complete identity with trypsin, since it does not digest casein and is not inhibited by other serine proteinase inhibitors.
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PMID:Cell-cell and cell-collagen interactions influence gelatinase production by human breast-carcinoma cell line 8701-BC. 755 30

The entire amino acid sequence of Ac1-Proteinase from Deinagkistrodon acutus venom was determined by using lysyl endopeptidase, metalloendopeptidase, trypsin and V8 protease. The hemorrhagic toxin had a typical zinc-chelating sequence His-Glu-X-X-His found in thermolysin.
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PMID:Primary structure of Ac1-proteinase from the venom of Deinagkistrodon acutus (hundred-pace snake) from Taiwan. 765 43

Proteinase inhibitor isolated from horsegram (Dolichos biflorus or Macrotyloma uniflorum) inhibited specifically the enzymes trypsin and chymotrypsin. The inhibitor contained seven disulfide linkages and was free from thiol groups. The inhibitor is resistant to denaturation by urea, guanidine hydrochloride or sodium dodecyl sulfate. Reduction of the inhibitor with dithiothreitol abolished both trypsin and chymotrypsin inhibitory activities. The kinetic plots of the reduction as followed by activity and loss in structure as reflected in the 257 nm CD band could be superposed; loss in the activity paralleled the loss in structure. The kinetics of the reduction process was complex; reduction of the inhibitor was slow and depended on the concentration of DTT. Reduction of the disulfide linkages with DTT affected the tertiary structure significantly and secondary structure was not affected considerably. Fluorescence quenching by acrylamide and potassium iodide suggested the unfolding of the molecule due to reduction. Thus, disulfide linkages play a predominant role in maintaining the three-dimensional structure of the inhibitor.
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PMID:Role of disulfide linkages in structure and activity of proteinase inhibitor from horsegram (Dolichos biflorus). 771 Oct 55

The principal digestive proteinases of the gypsy moth, Lymantria dispar, larval midgut were identified, and the subcellular distribution of the enzyme activities was determined. Proteinase activities of fifth-instar larvae were largely attributed to two luminal serine proteinases, a trypsin-like enzyme (TLE) and an elastase 2-like enzyme (ELA). TLE was purified to homegeneity by benzamidine-Sepharose affinity chromatography. With respect to size (M(r) = 25 kDa), substrate specificity, and interaction with trypsin inhibitors, the gypsy moth enzyme resembled mammalian pancreatic trypsin and trypsin-like enzymes from other insects. Gypsy moth elastase (ELA) was purified from the benzamidine-Sepharose flow-through by mono-Q FPLC. ELA exhibited a slightly smaller size (M(r) = 24 kDa) than TLE. The insect enzyme was inhibited by DFP and chymostatin but was unaffected by TPCK. ELA exhibited little esterolytic activity with BTEE. Succinyl-Ala-Ala-Pro-Leu p-nitroanilide was one of the best substrates for ELA, which is characteristic of elastase 2. TLE and ELA constituted c. 6% of the total soluble protein in midgut lumen of actively feeding fifth-instar larvae. Chymotrypsin and carboxypeptidase activities were not detected in any midgut fraction examined. The brush border membrane (BBM) leucine aminopeptidase (LAP) was isolated from CHAPS-solubilized BBM by FPLC. SDS-PAGE results indicated that the aminopeptidase has an apparent molecular size of c. 100 kDa. The aminopeptidase was inhibited by bestatin and was unaffected by serine proteinase inhibitors.
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PMID:Gypsy moth midgut proteinases: purification and characterization of luminal trypsin, elastase and the brush border membrane leucine aminopeptidase. 771 46

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in vertebrate brain. We have previously cloned cDNAs encoding two homologous kainate receptors (GFKAR alpha, 45 kDa, and GFKAR beta, 41 kDa) from goldfish brain and proposed a topology with three transmembrane domains (Wo, Z. G., and Oswald, R. E. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7154-7158). These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific antibodies and point and deletion mutations. We report here that the entire region between the previously proposed third and fourth transmembrane segments is translocated and likely to be extracellular in mature receptors. This was based on the following results. 1) The entire segment was protected from Proteinase K and trypsin digestion and could be immunoprecipitated by a site-specific antibody. 2) Functional sites for N-glycosylation are present in the C-terminal half of the segment, and 3) a mutation, constructed with an additional consensus site for N-glycosylation in the N-terminal half of the segment, was found to be glycosylated at that site. Given the fact that the N terminus of the protein is likely to be extracellular, this would place an even number of transmembrane segments between the extracellular N terminus and the glycosylated segment. In addition, results of N-glycosylation and proteolysis protection assays of GFKAR alpha mutations indicated that the previously proposed second transmembrane segment is not a true transmembrane domain. These results provide further evidence in support of a topology with three transmembrane domains that has important implications for the relationship of structure to function in ionotropic glutamate receptors.
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PMID:A topological analysis of goldfish kainate receptors predicts three transmembrane segments. 783 26

Inherent proteolytic activity was estimated in cockroach and basidiomycete extracts by quantifying acid soluble peptides that were released by incubating extracts with 1% bovine serum albumin as measured by Lowry (Sigma). Reference proteases released 740 (Proteinase K, 0.1 U), 248 (Trypsin, 1.0 U), and 533 micrograms/ml (Pronase, 0.5 U) of soluble peptides. American whole body cockroach extract (0.1 mg dry weight) released 330 micrograms/ml of soluble peptides, representing 13 trypsin equivalent units (TEU)/mg. Extracts from spores of the mushroom Pleurotus ostreatus released 230 micrograms/ml (0.9 TEU/mg) and Pleurotus cap extract released 112 micrograms/ml (0.5 TEU/mg). Mycelium of Pleurotus and the mushroom Psilocybe cubensis and spores of Psilocybe and the puffball Calvatia cyathiformis showed negligible amounts of proteolytic activity. The protease inhibitor phenylmethylsulfonyl flouride reduced the proteolytic activity of American whole body cockroach extract by 80% (@1 mM) and the inhibitor ethylene diaminetetraacetic acid inhibited the proteolytic activity of Pleurotus spores by 95% (@1 mM). Loss of allergen activity as determined by RAST inhibition and immunoprinting correlated with protease activity. Thus, in the preparation and handling of allergen extracts, one should employ conditions that minimize proteolysis.
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PMID:Protease activity in cockroach and basidiomycete allergen extracts. 822 32


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