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)

The active-site serine residue of Streptomyces griseus trypsin was converted to a cysteine residue, and the product, thioltrypsin, was purified through two chromatographic steps with organomercurial-Sepharose and soybean trypsin inhibitor-Sepharose as specific adsorbents. The purified preparation of thioltrypsin was found to contain a single residue of cysteine and to react with almost equimolar amounts of normality titrants. It exhibited only traces of catalytic activity toward typical trypsin substrates such as Nalpha-tosyl-L-arginine methyl ester, whereas it retained some activity toward "active ester" substrates such as Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester. The activity was inhibited by sulfhydryl-blocking reagents, but no inhibition was observed by reagents reactive with the active hydroxyl group of serine proteases. Leupeptin, a natural trypsin inhibitor of peptidyl nature, also inhibited thioltrypsin. Some difference in the mode of leupeptin inhibition, however, was detected between trypsin and thioltrypsin. The bindings of small synthetic ligands and soybean trypsin inhibitor to thioltrypsin were compared with those to trypsin.
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PMID:Thioltrypsin. Chemical transformation of the active-site serine residue of Streptomyces griseus trypsin to a cysteine residue. 41 Aug 3

The esterase activity of highly purified human urokinase on Nalpha-acetylglycl-L-lysine methyl ester is strongly inhibited by 1 X 10(-5) to 1 X 10(-2)M Cu++, Hg++, Ni++, Co++, Fe+++, and Mn++ solutions, whereas Na+, K+, Ca++, and Mg++ are weakly effective. This inhibition is parallel with the inhibition of activation of plasminogenby urokinase. There is no simple linear relation between inhibiton and concentration. Addition of ethylenediaminetetraacetate or electrodialysis fully reactivates the inhibited enzyme. These results are discussed in relation to similar effects of ions on trypsin.
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PMID:The effects of metal ions on esterase activities of urokinase. 41 20

Phenylalanine chloromethyl ketone covalently attached to porous glass beads was synthesized to serve as a solid-phase active site directed inhibitor of chymotrypsin-like proteolytic enzymes. The solid-phase reagent inhibited 20 nmol of bovine chymotrypsin per gram of glass and covalently bound 30 nmol of protein per gram of glass. Sepharose-bound lysine chloromethyl ketones were synthesized to serve as inhibitors of trypsin-like enzymes. Sepharose-MethionylLysyl chloromethyl ketone inactivated and bound about 6.8 nmol of enzyme per ml of settled gel. In a preliminary experiment, a cyanogen bromide cleavage of the methionine residues showed that it should be possible to release all peptides but the peptide containing the active-site histidine. The immobilized trypsin was also reduced, carboxymethylated and digested with chymotrypsin. The potential of the solid-phase approach is in the isolation of a specific serine proteinase and in the sequence determination of residues surrounding the active-site histidine.
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PMID:Solid-phase active site inhibitors of proteinases. 42 26

The amino acid sequences of human histones have been investigated for studies of histone evolution. The whole histone was prepared from human spleen and was separated into 3 fractions, H4+H3+H2A, H2B, and H1, by our technique of CM-cellulose chromatography. The H2B fraction was further purified by Bio-Gel P-60 chromatography. For sequence determination, the H2B molecule was first split into 4 major fragments I to IV, by limited chymotryptic digestion at pH 5.0 and 15 degrees C, followed by Sephadex G-50 chromatography. Fragments I and III were then digested with trypsin, yielding 18 and 16 peptides, respectively, on column and paper chromatographies. Sequence analyses of these tryptic peptides, as well as chymotryptic fragments II and IV, showed no differences from the corresponding parts of calf thymus H2B sequence, making it possible to locate fragments I to IV at residues 1--40, 41--42, 43--121 and 122--125 of the total sequence. The only new findings were microheterogeneities at residues 39 (75% valine and 25% isoleucine) and 124 (70% serine and 30% alanine). The sequence of the most basic cluster at residues 27--24, -Lys-Lys-Arg-Lys-Arg-Ser-Arg-Lys-, was confirmed with a peptide obtained from fragment I by staphylococcal protease digestion. Thus, it is concluded that the H2B sequence of lower mammals was conserved during the evolutionary process leading to man.
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PMID:Human spleen histone H2B. Isolation and amino acid sequence. 42 50

Insoluble elastin from copper-deficient animals has an amino acid composition intermediate between mature elastin and salt-soluble elastin (a higher lysine content and correspondingly low number of cross-links relative to the normal protein) and is solubilized by successive treatment with trypsin and chymotrypsin at 4 and 37 degrees C. Small amounts of B3H4 (11 mg--2 g of elastin) reduced allysine, allysine aldol, dehydronorleucine, and dehydromerodesmosine in insoluble elastin from copper-deficient pig aorta. In contrast, desmosine and isodesmosine were reduced only when a large excess of reductant (400 mg borohydride) was included in the reaction mixture. Reduction studies indicated that lysinonorleucine and merodesmosine were present in their dehydro forms to a greater extent in copper-deficient pig elastin than in normal elastin. After reduction with borohydride approximately 35% of the reduced form of the insoluble elastin remained insoluble after digestion with trypsin and chymotrypsin. A peptide containing the aldehyde oxidation product of lysine (allysine) and demonstrating an enrichment in glutamic acid was purified from the reduced form of copper-deficient pig elastin and partially sequenced. Its sequence (Gly-Ala-Glu-allysine-(Glu)...) and amino acid composition suggest: (1) clustering of glutamic acid residues in the elastin molecule, and (2) that allysine residues are not restricted to the alanine-enriched sites described for other elastin cross-links. Insoluble elastin from copper-deficient animals promises to be a useful tool for elastin sequence studies.
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PMID:Characterization of insoluble elastin from copper-deficient pigs. Its usefulness in elastin sequence studies. 42 11

A specific radioimmunoassay has been developed for human pancreatic cationic trypsin. The assay has been employed for the determination of immunoreactive forms of pancreatic cationic trypsin in blood. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone (TLCK) to prevent binding of the tracer to the serum inhibitors while maintaining its immunoreactivity. The average normal serum level determined was 26 ng/ml, with a range of 12--41 ng/ml. Eight of nine patients with acute pancreatic inflammation had at least a 15-fold elevation of total serum immunoreactive cationic trypsin. Cationic trypsinogen and cationic trypsin bound to alpha1-antitrypsin cross-react strongly in the radioimmunoassay. Thus it is possible to measure these potential molecular forms of cationic trypsin in serum. When normal human serum was fractionated on Sephadex G-200, all of the immunoreactive material eluted as a single peak of approximately 23,000 mol wt. No cationic trypsin could be detected in association with alpha1-antitrypsin or alpha2-macroglobulin. The 23,000-mol-wt peak was definitively shown to contain trypsinogen by affinity chromatography and by activation with human enteropeptidase. The identification of cationic trypsinogen in blood implies that the zymogen is secreted into the circulation by the pancreas rather than entering the bloodstream via absorption from the intestine.
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PMID:Determination of human pancreatic cationic trypsinogen in serum by radioimmunoassay. 43 51

The inactive 50,000-dalton fragment of human plasma alpha1-proteinase inhibitor resulting from limited proteolysis of the inhibitor by Crotalus adamanteus proteinase II has been isolated and partially characterized. The amino acid composition of the inactivated inhibitor indicates the loss of a peptide fragment from the intact inhibitor. Both intact and inactivated inhibitor contain COOH-terminal lysine. However, the NH2 terminus of the intact inhibitor is Glx, whereas that of inactivated inhibitor is methionine. NH2-terminal analysis of the inactive inhibitor fragment revealed the following sequence: -Met-Phe-Leu-Glu-Ala-Ile-Pro-Met-Ser-Ile-Pro-Pro-Gln-Val-Lys-Phe-Asn. The data show that the venom proteinase has inactivated alpha1- proteinase inhibitor by cleavage of a single bond which differs from that reported for trypsin or papain.
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PMID:Characterization of the inactive fragment resulting from limited proteolysis of human alpha1-proteinase inhibitor by Crotalus adamanteus proteinase II. 44 52

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.
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PMID:Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum. 45 24

An E-like abnormal hemoglobin was detected in a hematological patient and one of the members of her family. The composition of blood hemolyzates was characterized using acetate cellulose electrophoresis. The abnormal beta-chain was isolated by ion-exchange chromatography of total globin on CM-cellulose. Using peptide mapping of the abnormal beta-chain trypsin hydrolysates, it was shown that the amino acid substitution occurs in peptide beta T3. The amino acid analysis and determination of the abnormal fragment C-terminal amino acid allowed to establish the locus and type of this substitution. The first case of hemoglobin E(alpha 2 beta 2 26Glu leads to Lys) identification on the territory of the USSR is reported.
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PMID:[Primary structure of abnormal E-like hemoglobin]. 45 12

p-Chloromercuribenzoate-treated hemoglobin was digested by trypsin. The hydrolysate was subjected to gel-filtration on Bio-Gel P-4 and Sephadex G-50 columns, ion-exchange chromatography on CM-Sephadex and DE 52 columns, and paper electrophoresis. Peptides obtained by this procedure were analyzed for amino acid compositions and amino-terminal amino acid sequences. The results showed that p-chloromercuribenzoate-treated hemoglobin was hydrolyzed to a limited extent by trypsin at the bonds involving the carboxyl group of a lysine or arginine residue in planes A--E in the parent hemoglobin, which represent the external region of the parent tetramer. It is concluded therefore that the slight modification of hemoglobin enhances the susceptibility of the protein to proteases and that the hydrolysis of the modified protein is limited.
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PMID:Limited proteolysis of p-chloromercuribenzoate-dissociated hemoglobin by trypsin. 45 29


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