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)

Catalytic amounts of bovine beta-trypsin, bovine alpha-chymotrypsin and porcine plasmin establish a true thermodynamic equilibrium between virgin (I) (reactive site Lys15-Ala16 peptide bond intact) and modified (I) (this bond hydrolyzed) bovine trypsin/kallikrein inhibitor (Kunitz). The very slow reaction rates for attaining equilibrium are pH-dependent and differ for different enzymes. Optimal rates are for beta-trypsin at pH 3.75, for alpha-chymotrypsin at pH 5.5, and for plasmin at pH 5.0. Under conditions of optimum pH the equilibrium is reached with the highest rate by plasmin. In 10(-5)M inhibitor solutions the equilibrium concentrations of virgin and modified inhibitor are established by plasmin after almost 300 days starting from either pure virgin or pure modified inhibitor. Thus, the hydrolysis constant KHyd = [I]/[I] is determined to be 0.33 at pH 5.0. In spite of many unsuccessful attempts, this demonstrates that the reactive site peptide bond Lys15-Ala16 in the bovine trypsin inhibitor (Kunitz) can be hydrolyzed by catalytic amounts of endopeptidase. It further confirms that the hydrolyzed Lys15-Ala16 peptide bond in modified inhibitor is subject to thermodynamic control resynthesis.
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PMID:Hydrolysis-resynthesis equilibrium of the lysine-15--alanine-16 peptide bond in bovine trypsin inhibitor (Kunitz). 0 70

Three highly specific trypsin-like proteases from mouse submaxillary gland; nerve growth factor gamma subunit, beta nerve growth factor-endopeptidase, and epidermal growth factor-binding protein were tested for kallikrein activity. Low molecular weight kininogen was purified from mouse plasma and used as substrate for the three enzymes, and the kinin released by the enzymes was assayed by its ability to induce contraction of isolated rat uterus. All three enzymes were found to have significant kininogenase activity, and the most active enzyme, beta nerve growth factor-endopeptidase, has activity comparable to authentic kallikreins from other glandular sources. Essentially all of the kininogenase activity of submaxillary gland co-purifies with beta nerve growth factor-endopeptidase. Hence, beta nerve growth factor-endopeptidase appears to be identical with submaxillary gland kallikrein. Nerve growth factor gamma subunit, epidermal growth factor-binding protein, and beta nerve growth factor-endopeptidase have similar amino acid compositions and molecular weights, and are immunologically similar. Comparison of published partial primary sequence data confirms our conclusion that nerve growth factor gamma subunit, epidermal growth factor-binding protein, and kallikrein are very closely related enzymes. It is postulated that these three enzymes are members of a larger family of similar enzymes, all of which are involved in the processing of precursors to polypeptide hormones and growth factors.
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PMID:The relationship between glandular kallikrein and growth factor-processing proteases of mouse submaxillary gland. 11 Aug 5

In this paper, we present the amino-terminal sequence of rat tonin, an endopeptidase responsible for the conversion of angiotensinogen, the tetradecapeptide renin substrate, or angiotensin I to angiotensin II. It is shown that isoleucine and proline occupy the amino- and carboxy-terminal residues respectively. The N-terminal sequence analysis permitted the identification of 34 out of the first 40 residues of the single polypeptide chain composed of 272 amino acids. These results showed an extensive homology with the sequence of many serine proteases of the trypsin-chymotrypsin family. This information, coupled with the slow inhibition of tonin by diisopropylfluorophosphate, classified this enzyme as a selective endopeptidase of the active serine protease family.
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PMID:N-Terminal amino acid sequence of rat tonin: homology with serine proteases. 21 93

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.
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PMID:Protease II from Escherichia coli. Purification and characterization. 24 Aug 39

The biosynthesis of epidermal growth factor (EGF) was studied in mouse submaxillary glands incubated with L-[35S]cystine. EGF and EGF-like proteins were isolated from the gland homogenates by immunoprecipitation with anti-EGF antiserum. The major species appearing after short labeling periods is significantly larger (Mr, 9000) than EGF. The label in the Mr 9000 species plateaus after 1 hr whereas tha in EGF continuously increases. When glands are chased with unlabeled L-cystine after a brief period of labeling, the Mr 9000 peak decreases and a corresponding amount of label appears in EGF. The Mr 9000 species was isolated from boiled homogenates in which it accounts for approximately 1% of the total EGF content. It contains five of the six chymotryptic peptides of EGF and a sixth peptide which is a modified form of the COOH-terminal chymotryptic peptide of EGF. Of the arginyl esteropeptidases, gamma subunit of 7S nerve growth factor, beta-endopeptidase, trypsin, and EGF-binding protein, only the latter converts the isolated Mr 9000 species to EGF. The extrapeptide material released in the conversion comes from the COOH terminus of the Mr 9000 species. These results suggest that the Mr 9000 species is a biosynthetic precursor of EGF and that the EGF-binding protein is the specific intracellular cleaving enzyme that converts the precursor to EGF. In the process, the stable high molecular weight complex of EGF is formed.
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PMID:The biosynthetic precursor of epidermal growth factor and the mechanism of its processing. 31 39

A pancreatic endopeptidase localized to the beta-cells of the pancreas by immunohistochemical techniques has been purified to homogeneity by following its functional and antigenic characteristics as a glandular kallikrein (EC 3.4.21.8). The enzyme gave a single stained band on alkaline disc gel electrophoresis which corresponded in location with the kinin-generating activity eluted from a replicate gel, was of 54,000 molecular weight by gel filtration, was devoid of caseinolytic activity, elicited a monospecific antiserum in a rabbit, and gave a line of complete identity with a single constituent in pancreatic extract, crude urine, and purified urokallikrein when analyzed with monospecific antibody to urokallikrein. The pancreatic glandular kallikrein generated three cleavage products of increasing anodal mobility from bovine and porcine proinsulin, and the presence of pancreatic kininase or bovine carboxypeptidase B increased the quantity of these products. Although the conversion products did not correspond to diarginyl- and monoarginylinsulin, the product of intermediate mobility was also obtained when proinsulin was treated with a low concentration of trypsin in the presence of kininase. The most rapidly migrating product did correspond to desalanylinsulin in the reference standard. Kininase alone had no action on proinsulin, and aprotinin prevented cleavage by kallikrein alone or in combination with kininase. Although the chemical structure of the proinsulin cleavage products has not been established, human pancreatic kallikrein is considered a putative activator of proinsulin because of its location in the beta-cell, its preferential action on proinsulin and kininogen as compared to azocasein, and its capacity to generate insulin intermediate products that are further modified by human pancreatic kininase or bovine carboxypeptidase B.
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PMID:Sequential cleavage of proinsulin by human pancreatic kallikrein and a human pancreatic kininase. 38 42

The proteolytic activity of the extracellular protease of Serratia marcescens was compared with that of trypsin on N, N-dimethyl casein. The peptides produced from exhaustive hydrolysis of alpha casein by the protease and by trypsin were of similar size as measured by gel filtration on P-10 Agarose. We conclude that the protease of S. marcescens in an endopeptidase with trypsin-like activity on proteins, producing oligopeptides. End group analysis of the peptides formed by the S. marcescens protease suggests that the protease has a unique substrate specificity, hydrolyzing only a peptide bond whose carboxyl group is donated by proline. The protease was inactive on the synthetic peptides with proline donating the carboxyl group, but hydrolyzed various types of natural proteins. Its narrow and novel substrate specificity makes this enzyme a potential tool for the determination of the primary structure of proteins.
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PMID:The extracellular metalloprotease of Serratia marcescens. 2. Comparison with trypsin and substrate specificity. 79 98

This study demonstrates that a serine endopeptidase of pancreatic origin (elastase 2) circulates in human blood. A specific and highly sensitive radioimmunoassay has been developed for pancreatic elastase 2 in human serum. The inactivation of elastase 2 employed as radioiodinated tracer with an active site-specific reagent (phenylmethanesulfonyl fluoride) was necessary to prevent its binding by serum alpha1-antitrypsin and alpha2-macroglobulin while maintaining its immunoreactivity. The assay is based upon competition of standard human pancreatic elastase 2 with 125I-labeled phenylmethanesulfonyl elastase 2 for specific antibody binding sites, after which a second antibody precipitation step is used to separate bound from free 125I-labeled phenylmethanesulfonyl elastase 2. The minimum detectable concentration of elastase 2 was 0.9 ng/ml. The average normal fasting serum level determined was 71 ng/ml, approximately 80-fold greater than the minimum detectable amount. The form of radioimmunoassayable elastase 2 in normal human serum has been investigated by gel filtration of serum samples on Sephadex G-200 followed by radioimmunoassay of column fractions. The majority of the immunoreactive elastase 2 is eluted from G-200 in the void volume. While a minor amount of elastase 2 is eluted in a position consistent with alpha1-antitrypsin-elastase 2 complex, no free elastase or free proelastase is detectable. Addition of exogenous elastase 2 to normal serum prior to gel filtration on G-200 produced an increase only in the peak of radioimmunoassayable elastase bound to alpha1-antitrypsin. In vitro experiments have demonstrated that while elastase 2 bound to alpha1-antitrypsin is immunologically reactive, alpha2-macroglobulin-bound elastase 2 cross-reacts less than 2% in this radioimmunoassay. The assay has been shown to be specific for elastase 2. Human pancreatic elastase 1, anionic trypsin, chymotrypsin I, and chymotrypsin II do not cross-react in this assay system. The major advantages of this radioimmunoassay over enzymatic assays are its high sensitivity and ability to measure the enzyme in terms of its total protein concentration.
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PMID:Pancreatic elastase in human serum. Determination by radioimmunoassay. 83 29

After enzymatic digestion of chicken myoglobin by trypsin, chymotrypsin or thermolysin, the separation of peptides was performed by column chromatography on various ion exchange resins. Each peptide was purified by high-voltage paper electrophoresis or by chromatography either on paper or on ion-exchange resin, and its complete amino acid sequence was then determined by the combined dansyl-Edman procedure and by endopeptidase digestions. The whole globin was submitted to automatic Edman degradation using the Beckman sequencer. Residues have been positioned from overlaps of sequence data between tryptic (T), chymotryptic (C) and thermolysin (Th) peptides. The stepwise degradation of the whole globin confirmed the alignment of the N-terminal third of the molecule. The combination of these different approaches has led to the complete determination of the 153 residues sequence forming the polypeptide chain of chicken myoglobin. Comparison of the established chicken myoglobin structure with those from other species shows a conservation of structure, although the avian protein exhibits more variations in its amino acid sequence than has been found between other known myoglobins which all belong to mammalian species.
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PMID:The primary sequence of chicken myoglobin (Gallus gallus). 116 72

Leupeptin is a small peptide microbially derived inhibitor of certain proteolytic enzymes. Using N-alpha-benzoyl-DL-arginine 4-nitroanilide as substrate, we found a novel leupeptin-sensitive proteolytic enzyme in N-methyl-N-nitrosourea(MNU)-induced rat mammary adenocarcinoma. This enzyme was apparently different from urokinase-type plasminogen activator or cathepsin B and was present in mammary tumour at levels at least 20 times higher than those in normal mammary tissue. This enzyme was separated and purified from crude extracts of MNU-induced mammary adenocarcinoma approx. 1900-fold with 34% yield. It was a trypsin-like serine endopeptidase and had a pH optimum at 7.0. The native enzyme had an apparent M(r) of 180,000 and exhibited four isoelectric points ranging from 4.3 to 5.0. Electrophoresis of denatured enzyme, however, yielded, with reduction, a major band with an apparent M(r) of 37,500 and a minor band with an apparent M(r) of 35,500. The N-terminal 23 residues of the major band were Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Xaa12-Pro13- Val14- Gln15-Val16-Xaa17-Leu18-Xaa19-Val20- Trp21-Leu22-Pro23. These and other properties of this enzyme suggested that it most closely resembles rat skin tryptase, followed by rat peritoneal mast-cell tryptase and then by tryptases from other species. The rat, like human and mouse, may carry multiple tryptase genes, and this mammary-tumour enzyme may be an additional form of rat tryptase within a new serine-proteinase family.
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PMID:Separation, purification and N-terminal sequence analysis of a novel leupeptin-sensitive serine endopeptidase present in chemically induced rat mammary tumour. 131 62


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