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)

Two thiol-activated endopeptidases with pH optima near pH 7.5 were isolated from the supernatant fraction of rabbit brain homogenates by DEAE-cellulose chromatography, gel filtration and isoelectrofocusing. Peptide bond hydrolysis was measured quantitatively by ion-exchange chromatography with an amino acid analyzer. Brain kininase A hydrolyzes the Phe5-Ser6 peptide bond in bradykinin (Bk), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9. It is isoelectric near pH 5.2 and has a molecular weight of approximately 71 000. The enzyme also hydrolyzes the Phe-Ser peptide bond in Lys-Bk, Met-Lys-Bk, des-Arg1-Bk, Lys9-Bk, Pro-Gly-Phe-Ser-Pro-Phe-Arg, and Gly-Pro-Phe-Ser-Pro-Phe-Arg, but does not hydrolyze (0.1%) this bond in des-Phe8-Arg9-Bk. Brain kininase B hydrolyzes the Pro7-Phe8 peptide bond in Bk. It is isoelectric at pH 4.9 and has a molecular weight of approximately 68 000. Brain kininase B also hydrolyzes the Pro-Phe bond in Lys-Bk, Met-Lys-Bk, Lys9-Bk, Ser-Pro-Phe-Arg, and Phe-Ser-Pro-Arg. Pretreatment of denatured kininogen with brain kininase A or B did not reduce the amount of trypsin-releasable Bk from this precursor protein, indicating that the Bk sequence, when part of a large protein, is not a substrate for either enzyme. However, kininase A and B hydrolyze the octadecapeptide Gly-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gin-Val. The data show that a large part of the C-terminal portion of bradykinin is important for the brain kininase A activity and, for both enzymes, the size of the peptide and presumably the residues adjacent to the scissle bond are important in determining the rate of peptide bond hydrolysis by these endopeptidases.
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PMID:Isolation of brain endopeptidases: influence of size and sequence of substrates structurally related to bradykinin. 0 20

Some properties of rat skin benzoylarginine-2-naphthylamide hydrolase types I (preparations I and AI) and II (preparations II and NII) were studied. Both types were activated by dithiothreitol and EDTA, but responded differently to 1 mM KCN, when benzoylarginine-2-naphthylamide (BANA) was used as a substrate: type I was inhibited, while type II was activated. When leucine-2-naphthylamide was used as a substrate, both types were activated by KCN. Thiol proteinase inhibiting substances, like heavy metals, iodoacetic acid, 4-chloromercuribenzoic acid, and tosyllysine chloromethylketone, inhibited the enzymes. Diisopropylfluorophosphate, phenylmethylsulfonyfluoride, 4-aminobenzamidine, and high-molecular-weight trypsin inhibitors were without effect. The substrate specificity of rat skin BANA hydrolase resembled that of an amino acid naphthylamidase, naphthylamides of methionine, lysine, arginine, and alanine being hydrolyzed most rapidly. The rate of hydrolysis of BANA was only 11% of that of methionine naphthylamide. Amino acid esters with a free alpha-amino group were also good substrates. The transformation of type II to type I at acidic pH was studied. During the transformation amino acids or peptides were formed and probably some inhibitor present in type II was destroyed proteolytically.
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PMID:Alpha-N-Benzoylarginine-2-naphthylamide hydrolase (cathepsin BI?) from rat skin. III. Substrate specificity, modifier characteristics, and transformation of the enzyme at acidic pH. 0 11

Three new protease inhibitors were isolated and purified about 200-fold from hemolymph of silkworm larvae, Bombyx mori, using ion-exchange and affinity chromatography. Two of the three inhibitors were basic proteins (SCI-I had pI 9.4 and SCI-II had pI 9.6) and one was acidic (SCI-III had pI 4.0). The molecular weight of each inhibitor was determined to be 7,000 by the sedimentation equilibrium method. The amino acid composition of the inhibitors were similar except for the contents of Asp, Glu, Ile, Leu, and Lys. Val, His, and Trp were not present in the inhibitors and Met appeared only in SCI-III. The CD spectra of the inhibitors were all similar and indicated a low content of alpha-helical structure (10% at most). Each inhibitor could inhibit the protease and esterase activities of bovine alpha-chymotrypsin at a one-to-one molar ratio, and the dissociation constants were 3.1 X 10(-9)M for SCI-I and II and 1.3 X 10(-8)M for SCI-III. Only SCI-II showed a weak inhibitory activity against bovine trypsin. Subtilisin BPN' and papain were not inhibited by these inhibitors.
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PMID:Chymotrypsin inhibitors from hemolymph of the silkworm, Bombyx mori. 2 86

The trypsin and chymotrypsin inhibitor from chick peas (CI) is stable in HCl 0.001 M -- 0.01 M and in KOH 0.01 M -- 0.05 M even after 24 h. Increased KOH concentrations decrease considerably the inhibitory activity already after 1 h. Maleyation and succinylation of the inhibitor resulted in almost full loss of its trypsin-inhibitory activity but had no effect on the chymotrypsin-inhibitory activity. A series of modifications directed towards tyrosyl residues showed that iodination influenced only the chymotrypsin-inhibitory activity; however, nitration and arsanilation affected not only the chymotrypsin-inhibitory activity but also the trypsin-inhibitory activity. Treatment of the inhibitor with CNBr and chloramine T resulted only in a decrease in the chymotrypsin-inhibitory activity indicating that the only methionine is involved in the chymotrypsin-inhibitory activity. When CI-fragment A, previously treated with trypsin at pH 3.75, was further treated with carboxypeptidase B, a release of three lysyl residues per mole protein was found. CI was separated by equilibrium chromatography on SP-Sephadex column into two isoinhibitors, CII and CIII, respectively. Both inhibited trypsin and chymotrypsin with the same specific activity as CI. They differed from each other only in a glutamyl, aspartyl, glycyl and alanyl residue.
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PMID:Trypsin and chymotrypsin inhibitor from chick peas. Selective chemical modifications of the inhibitor and isolation of two isoinhibitors. 4 22

Five protease inhibitors, I--V, in the molecular weight range 7000--8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I--V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibit chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residued of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.
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PMID:Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean. 7 87

The mouse mammary tumor virus (MuMTV) contains several low-molecular-weight proteins which, together with the genomic RNA, constitute the core structure of the virion. The most abundant protein in the core is the 27,000-dalton protein (p27), and, by analogy to the type C viruses, this protein probably forms the core shell. In mouse mammary tumor cell lines (GR and Mm5MT) producing MuMTV the major p57 antigenic specificity resides in a large protein, which migrates in polyacrylamide gels as a doublet of 77,000 and 75,000 daltons (p 77/75). A series of lower-molecular-weight proteins, p61, p48, p38, and p34, is also present in small amounts and is probably derived by proteolytic cleavage of the p 77/75. These proteins have been identified by immunoprecipitation with monospecific antiserum, and their sequence relatedness to p27 has been determined by an analysis of the peptides after trypsin digestion. After a 15-min pulse with [35S]-methionine, all of the p27-related proteins in these cell lines were labelled and, during a subsequent chase, progressively disappeared. The p27 was labeled poorly during the pulse, but the amount of label in this protein increased during the chase. A quantitation of these experiments suggested that the majority of the p27-related proteins were quite rapidly turned over in these cell lines. Hence, if p27 is derived by a progressive proteolytic cleavage mechanism, then the process is inefficient in the GR cells and only moderately efficient in the Mm5MT cells. When MuMTV was isolated from the culture medium of these cells harvested at 5-min intervals, the major p27-related protein was p34. The p27 accounted for only 29% of the anti-p27 serum immunoprecipitable proteins compared to 95% in virus isolated from an 18-h harvest. Incubation of the rapid-harvest virus at 37 degrees C for 2 h resulted in some conversion of p34 to p27. These results suggest that some of the p27 in MuMTV is formed in the virions by proteolytic cleavage of p34.
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PMID:Polyproteins related to the major core protein of mouse mammary tumor virus. 7 87

A preparation rich in basement membranes isolated from rat testes (STBM) was exposed to pepsin, collagenase, trypsin, and pronase to obtain soluble fractions. The immunological reactivity of these fractions was studied by gel immunodiffusion or by passive hemagglutination tests against an anti-STBM serum. All fractions reacted with the antiserum, but the highest titer was detected when the antiserum was reacted with a fraction that contained only traces of hydroxyproline (fraction 1), whereas low titers were obtained with collagen or collagen fragments isolated from STBM. Antibodies in the anti-STBM serum were mainly directed to the glycoproteins of STBM not related to collagen. Fraction 1, obtained by subsequent collagenase and trypsin digestion of STBM and purification by Sephadex G-200, was a high molecular weight glycoprotein that was free of half-cystine and methionine, had only traces of hydroxyproline, and contained 7.2% neutral sugars, 0.26% sialic acid, and 8.7 residues of glucosamine per 1000 residues of amino acids.
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PMID:Isolation and immunological reactivity of soluble fractions from rat seminiferous tubule basement membrane. 9 Apr 90

A surface coat of host serum proteins was detected on virulent Treponema pallidum by sodium dodecyl sulfate-gel electrophoresis. The loosely associated serum proteins could be removed by repeated washings in a protein-free medium. Washed T. pallidum retained the ability to readsorb numerous host proteins from rabbit serum as well as iodinated rabbit or human albumin. In addition, various avidly associated host serum proteins including albumin, alpha(2)-macroglobulin, transferrin, ceruloplasmin, immunoglobulin G, immunoglobulin M, and C3 were identified on the outer envelope of washed treponemes by an immunoadsorbent technique with protein A-bearing staphylococcus. Hyaluronidase treatment did not remove the avidly associated host proteins from the surface of washed treponemes, whereas trypsin treatment resulted in decreased levels of agglutination. Electrophoretic patterns of trypsin-treated treponemes showed that treponemal proteins as well as adsorbed host proteins were released concurrently by protease digestion. Reacquisition studies involving alpha(2)-macroglobulin and transferrin suggested the presence of noncompetitive binding sites for serum proteins on the treponemal outer envelope. Finally, differences among the T. pallidum preparations from individual rabbits with respect to incorporation of [(35)S]methionine, extent of agglutination with antisera, and length of time required for removal of avidly associated host proteins by trypsin treatment indicated biological variability among the treponemal populations.
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PMID:Surface-associated host proteins on virulent Treponema pallidum. 9 74

Highly purified basic proteins have been isolated from bovine and turkey brains by a novel method employing acid-acetone extraction. The final product gave a single band on polyacrylamide gel electrophoresis at pH 4.3 and in the presence of sodium dodecyl sulfate. Both proteins have arginine at the COOH-terminus while the NH2-terminal residue cannot be detected and is probably blocked. A higher ratio of histidine to lysine and a greater proportion of serine and valine was found for the turkey compared with the bovine protein. Both proteins contain one tryptophan and two methionine residues. However, it was found from cyanogen bromide treatment that there is a marked difference in the location of one of the methionine residues, while the tryptophan-containing peptides liberated after trypsin digestion have different mobilities on peptide maps. When dissolved in water these proteins give a typical random coil curve from circular dichroism (CD), whereas in 80% methyl alcohol they assume a 25% alpha-helix.
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PMID:Characterization of turkey myelin basic protein isolated by a simple procedure. 9 40

1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500.
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PMID:Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZ beta-galactosidase of Escherichia coli by beta-D-galactopyranosylmethyl-p-nitrophenyltriazene. 10 21


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