Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A chymotrypsin-like enzyme (EC 3.4.21.-) was purified from granules of human neutrophiles (polymorphonuclear leucocytes). The isolation procedure included differential salt extractions of the granules followed by affinity chromatography on 4-phenylbutylamine-Affi-Gel. This rapid purification method resulted in obtaining pure enzyme in relatively high yield in short time. The purified granulocyte chymotrypsin-like enzyme has a minimum Mr of 22 378, calculated from its amino acid composition. The Mr value obtained by sodium dodecyl sulphate gel electrophoresis was 20 000-23 000. The enzyme did not react with antibodies which are monospecific to granulocyte elastase. The granulocyte chymotrypsin-like enzyme was inactivated by Dip-F and by the chloromethyl ketone derivatives Z-PheCH2Cl and Z-(Gly)2-PheCH2Cl but not by Tos-PheCH2Cl. It therefore appears that the enzyme has serine and histidine side chains in its active site, like pancreatic chymotrypsin. The granulocyte enzyme substrate specificity is similar to that of pac-Tyr-Nan and Ac-Phe-1-ONap. It also has an intrinsic weak hydrolytic activity towards some classical elastase substrates such as Boc-Ala-ONp and Ac-DL-Ala-1-ONap. The granulocyte enzyme is inhibited by human serum and by human alpha1-antitrypsin. Its affinity for alpha1-antitrypsin is weaker than that of granulocyte elastase for the same inhibitor. The enzyme is stable at neutral pH at 37 degrees C, but unstable at pH 3.5 and at elevated temperature.
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PMID:A rapid method for purification of human granulocyte cationic neutral proteases: purification and characterization of human granulocyte chymotrypsin-like enzyme. 108 Oct 3

The major coat protein of bacteriophage f1 radioactively labeled with specific amino acids was solubilized with deoxycholate and digested with trypsin or alpha-chymotrypsin. The degree of proteolysis of the coat protein was assayed by gel filtration chromatography of the digest in the presence of deoxycholate. Hydrolysis occurred at residues in the hydrophilic termini of the coat, releasing peptides containing proline, lysine, and phenylalanine. No cleavage occurred at the tyrosine or methionine residues in the hydrophobic core. However, chymotrypsin could cleave somewhat at these residues in the absence of deoxycholate. A model for the topography of the micellar complex of coat protein and deoxycholate is presented in which the hydrophobic sequence of the coat is bound to deoxycholate within a micelle, while the hydrophilic termini of the coat project from the micelle.
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PMID:Proteolytic digestion of the micellar complex of f1 coat protein and deoxycholate. 112 54

1. RNAase (ribonuclease) U2, a purine-specific RNAase, was reduced, aminoethylated and hydrolysed with trypsin, chymotrypsin and thermolysin. On the basis of the analyses of the resulting peptides, the complete amino acid sequence of RNAase U2 was determined, 2. When the sequence was compared with the amino acid sequence of RNAase T1 (EC 3.1.4.8), the following regions were found to be similar in the two enzymes; Tyr-Pro-His-Gln-Tyr (38-42) in RNAase U2 and Tyr-Pro-His-Lys-Tyr (38-42) in RNAase T1, Glu-Phe-Pro-Leu-Val (61-65) in RNAase U2 and Glu-Trp-Pro-Ile-Leu (58-62) in RNAase T1, Asp-Arg-Val-Ile-Tyr-Gln (83-88) in RNAase U2 and Asp-Arg-Val-Phe-Asn (76-81) in RNAase T1 and Val-Thr-His-Thr-Gly-Ala (98-103) in RNAase U2 and Ile-Thr-His-Thr-Gly-Ala (90-95) in RNAase T1. All of the amino acid residues, histidine-40, glutamate-58, arginine-77 and histidine-92, which were found to play a crucial role in the biological activity of RNAase T1, were included in the regions cited here. 3. Detailed evidence for the amino acid sequence of the sequence of the proteins has been deposited as Supplementary Publication SUP 50041 (33 PAGES) AT THE British Library (Lending Division)(formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975), 145, 5.
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PMID:The amino acid sequence of ribonuclease U2 from Ustilago sphaerogena. 115 64

The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.
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PMID:Calcium-binding protein of bovine intestine. The complete amino acid sequence. 117 41

The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.
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PMID:The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver. 118 Aug 94

Two alkaline proteases, one splitting preferentially the substrates of chymotrypsin (ATEE) and the other one those of trypsin (BAEE), were separated and partially purified by chromatographic means from human skin extract made in a buffer containing 1.07 mol/1 KC1. The proteins soluble in dilute buffer were removed by a prior extraction. The enzymes could be separated effectively only in the presence of KC1 at a high conc-ntration since large molecular size aggregates or polymers were formed in solutions of low ionic strength. In the presence of 2 mol/1 KC1 the molecular size of the BAEE-hydrolysing enzyme was 120000 and that of the ATEE-hydrolysing enzyme 30000. The ATEE-hydrolysing enzyme was purified by Sephadex G-100 gel filtration and DEAE-cellulose chromatography about 250 fold. It also hydrolysed esters of tryptophane and phenylalanine as well as casein with optimum pH 7.8--8.2. The enzyme was inhibited effectively by LBTI, SBTI and partially by trasylol, TPCK and TLCK, but not by E-600 and SH-modifers. The hydrolysis of ATEE was doubled in the presence of 1 mol/lKCl, NaCl, KBr or NaBr but that of casein was inhibited to some extent. Human serum and alpha-1-antitrypsin inhibited this enzyme but not C1-inactivator. alpha-2-Macroglobulin did not protect if from inhibition by SBTI. The BAEE-hydrolysing enzyme was purified by Sephadex G-100 gel filtration and hydroxylapatite chromatography about 30 fold. It also split other esters of substituted basic amino acids as well as BAPA and histone proteins with optimum pH 7.5--8.2. It was inhibited by Trasylol and TLCK, but not by LBTI, SBTI, OMTI, TPCK, E-600, SH-modifiers, human serum, C1-inactivator or alpha-1-antitrypsin. Neither of these enzymes is exactly similar to any one of the enzymes so far separated from human tissues or fluids.
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PMID:Human skin proteases: separation and characterization of two alkaline proteases, one splitting trypsin and the other chymotrypsin substrates. 120 Jul 4

1. The reactivity of alpha-chymotrypsin toward p-nitrophenylacetate has been studied in dimethylformamide, dimethylsulfoxide, formamide and methylacetamide. p-Nitrophenol is liberated in dimethylsulfoxide only. 2. The reactions of alpha-chymotrypsin in dimethylsulfoxide are characterized by the same kinetic and equilibrium constants with either the p-nitrophenyl esters of straight chain carboxylic acids (from acetic to n-caprylic) or with the "specific substrate", N-carbobenzoxy-DL-phenylalanine p-nitrophenyl ester. This signifies that reactions of alpha-chymotrypsin in dimethylsulfoxide, unlike those in aqueous medium, have no specificity toward su-strate structure. 3. The stoichiometry of alpha-chymotrypsin reactions in dimethylsulfoxide was shown to be about five moles of substrate per mole of enzyme. After attaining this stoichiometry, the reaction is completed. 4. Optical rotatory dispersion spectra indicate that in non-aqueous media alpha-chymotrypsin undergoes a large conformational transition which results in a random coil. 5. Chymotrypsinogen, trypsin, trysinogen, lysozyme and serum albumin react with p-nitrophenylacetate in dimethylsulfoxide at rates which are approximately equal to those of alpha-chymotrypsin. Thus, the "activity" of alpha-chymotrypsin in dimethylsulfoxide toward p-nitrophenylacetate does not differ from the "activity" of other proteins, some of which are not even hydrolytic enzymes.
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PMID:The reactions of alpha-chymotrypsin and related proteins with ester substrates in non-aqueous solvents. 120 14

Two bitter peptides, H-Phe-Tyr-Pro-Glu-Leu-Phe-OH (I) and H-Val-Glu-Val-Phe-Ala-Pro-Pro-Phe-OH (II) were isolated from casein, hydrolyzed by alpha-chymotrypsin. The hexapeptide is cleaved by thermolysine between Glu and Leu. The two fragments are bitter too. A bitter dodecapeptide (III) was obtained 20 min hydrolysis of casein with trypsin. On account of amino acid composition and N-terminus peptide III is probably identical with a peptide from a 12 hrs hydrolyzate, described in 1970 by Matoba. The peptides I and III have equal taste tresholds in the range of 0.08-0.10 muM/ml.
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PMID:[Bitter peptides of casein isolated by hydrolysis with alpha-chymotrypsin and trypsin (author's transl)]. 122 11

A variety of azobenzene compounds having bis-quaternary nitrogens have been shown to accelerate the hydrolysis by chymotrypsin of certain specific substrates by an allosteric mechanism. One of the most potent, 2,2'-bis[alpha-(benzyldimethylammonium)methyl]azobenzene dibromide (2,2'-QBzl) accelerated the hydrolysis of glutaryl-L-phenylalanine p-nitroanilide 40-fold at saturating concentration. Acceleration was by increasing kcat without altering Km. The hydrolysis of acetyl-L-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide was also accelerated by Q-Bzl (25-fold and 1.8-fold respectively) while the hydrolysis of hemoglobin, azocoll and a number of esters was not affected. The inactivation of chymotrypsin by diphenylcarbamyl chloride and diphenylcarbamyl fluoride was accelerated by 2,2'-Q-Bzl. Reac;ivation in the presence of NH2OH was also accelerated, but in the absence of added nucleophile (i.e. of NH20H) no increase in rate was detectable. An allosteric effector was covalently attached to chymotrypsinogen A by reaction with 2,2'-bis[alpha-(o-bromomethylbenzyldimethylammonium)methyl]azobenezene dibromide. The product, when converted to active enzyme, was about 4 times more active than chymotrypsin as a result of an increase in kcat of hydrolysis; Km was unaffected. The mechanism of the allosteric acceleration process is not known but, because for all of the substrates affected acylation of the enzyme is rate-limitimg, it is tentatively suggested that the effectors facilitate proton transfer to the leaving group by an inductive effect on the 'charge relay system'. Spectral studies indicate that the allosteric site is a portion of the enzyme with a polarity near that of water, possibly on the outside surface of the enzyme molecule.
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PMID:Allosteric activation of the hydrolysis of specific substrates by chymotrypsin. 124 86

The complete amino acid sequence of rat thyrocalcitonin has been determined by automated Edman degradations of the intact molecule, a cyanogen bromide fragment, and by degradations of mixtures of peptides produced by hydrolysis of the hormone with trypsin and chymotrypsin. The sequence determined was H2N-Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Leu-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ser-Ile-Gly-Val-Gly-Ala-Pro-NH2. This sequence differs in only two positions from that found in the human hormone, i.e. leucine-16 in the rat vs phenylalanine-16 in the human, and serine-26 in the rat vs alanine-26 in the human. These similarities and differences are consistent with the previously reported immunological properties of the hormones isolated from these two species.
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PMID:The complete amino-acid sequence of rat thyrocalcitonin. 127 75


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