Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q99895 (chymotrypsin)
 9,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In bovine protein C normal activation by the thrombin-thrombomodulin complex requires binding of calcium to one high affinity binding site, contained in a protein fragment that lacks the gamma-carboxyglutamic acid (Gla) region (Esmon, N. L., De Bault, L. E., and Esmon, C. T. (1983) J. Biol. Chem. 258, 5548-5553). In this work, the calcium binding to and the conformational change induced by calcium in the corresponding Gla-domainless fragment of bovine factor X, prepared by limited proteolysis by chymotrypsin, were compared with the calcium-binding properties of Gla-domainless protein C. Equilibrium dialysis experiments demonstrated that the proteolytically modified factor X has one high affinity calcium ion-binding site with Kd = 180 microM, a value almost identical to the Kd for the binding of calcium to proteolytically modified protein C. Measurements of the rate of disulfide bond reduction by thioredoxin showed that the disulfide bonds of both factor X and protein C lacking the Gla domains were more rapidly reduced in the absence than in the presence of calcium. Thus, calcium binding induces a conformational change in both proteolytically modified proteins. Calcium binding to Gla-domainless protein C is accompanied by a quenching of the intrinsic tryptophan fluorescence and by changes in the CD spectrum, indicative of perturbation of the environment of aromatic amino acids by the metal ion. However, no such changes were observed with the proteolytically modified factor X. This difference may be due to the fact that one tryptophan residue (in position 84) is present in the light chain of the proteolytically modified protein C but none in the light chain of the modified factor X. The light chain of factor X has beta-hydroxyaspartic acid in position 64 which is homologous to the beta-hydroxyaspartic acid in position 71 in the light chain of protein C. Our results are compatible with the hypothesis that beta-hydroxyaspartic acid is involved in the Ca2+ ion binding.
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PMID:Calcium-binding properties of bovine factor X lacking the gamma-carboxyglutamic acid-containing region. 654 30

The polypeptide molecular weight of lecithin-cholesterol acyltransferase (LCAT) (45000) was obtained by deducting the weight of carbohydrate moiety (25%, w/w) from the total molecular weight of 60000. LCAT was found to have a relatively high content of glutamic acid, aspartic acid, glycine, and leucine residues and four half-cystines. The carbohydrate content was found to be about 25% (w/w): hexoses, 13%; hexosamines, 6.2%; and sialic acid, 5.4%. The total number of 408 amino acid residues per mole and the mean residue weight of 110.3 were found. From fluorescence spectroscopy analysis, 6-7 mol of tryptophan were found per mole of LCAT in 10 mM phosphate (pH 7.4). However, when LCAT was digested by the mixture of chymotrypsin and pronase the tryptophan residues increased to 10-11 mol/mol of LCAT, which agrees well with data obtained previously by ultraviolet absorption spectroscopy. A partial specific volume of 0.707 mL/g was determined by compositional analysis. Human LCAT was found to have a relatively high extinction coefficient (E1%1cm) of 21 at 280 nm and neutral pH. Two residues of cysteine per mole of LCAT were estimated both in the presence or absence of sodium dodecyl sulfate by titration with 5,5'-dithiobis-2-nitrobenzoic acid. The enzyme showed a lower tendency to staining with Coomassie blue R-250 than bovine serum albumin. The enzyme was rapidly inactivated by diisopropyl fluorophosphate (DFP), regardless of whether the free sulfhydryl were blocked or not. The enzyme was also irreversibly inhibited by cysteine above concentrations of 1 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of lecithin-cholesterol acyltransferase from human plasma. 3. Chemical properties of the enzyme. 662 99

The complete amino acid sequence of the collagen-binding domain of bovine plasma fibronectin has been determined. The fragment, generated by digestion of fibronectin with plasmin and chymotrypsin, contains 340 residues (260-599 of fibronectin) with threonine and tryptophan as the amino-terminal and carboxyl-terminal amino acids, respectively. 24 half-cystines and no cysteines are present in the sequence. Three glucosamine-based oligosaccharide groups are attached to Asn-399, Asn-497 and to Asn-511, respectively. Two of the three types (I and II) [Petersen et al. (1983) Proc. Natl Acad. Sci. USA 80, 137-141] of internal homology occur in the fragment, namely four of the at least twelve stretches of type I sequence homology, 'fingers', and two stretches of type II homology. The type I homology is present in two other plasmic fragments from fibronectin, while the type II homology has been found in the collagen-binding domain only.
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PMID:Complete primary structure of the collagen-binding domain of bovine fibronectin. 671 32

A double selection method for isolating active enzyme molecules, using substrate analog affinity chromatography and elution with transition state analogs, is described. To demonstrate the principle, a mixture containing native chymotrypsin and [3H]deoxychymotrypsin, in which the active site serine had been converted to [3H]alanine, was applied to a column containing immobilized D-tryptophan methyl ester. Both forms of chymotrypsin were retained. Catalytically active enzyme was selectively desorbed with the peptide aldehyde chymostatin, leaving catalytically inactive deoxychymotrypsin bound to the substrate analog affinity column. This affinity technique may afford a simple and general method for separating enzymes and other catalysts according to their molecular turnover numbers.
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PMID:Transition state affinity jump chromatography. A double selection method for isolating catalytically active enzymes and other molecules. 677 75

Ozone decreased the trypsin, chymotrypsin, and elastase inhibitory activities of human alpha 1-proteinase inhibitor (alpha 1-PI) both in plasma and in solutions of the pure inhibitor. The total loss of porcine elastase inhibitory activity required 18 mol of ozone/mol of pure alpha 1-PI and approximately 850 mol of ozone/mol of alpha 1-PI in plasma. A corresponding loss of the ability to inhibit human leukocyte elastase was observed. Inactivated alpha 1-PI contains four residues of methionine sulfoxide, in addition to oxidized tyrosine and tryptophan. Electrophoretic analysis demonstrated that the ozone-inactivated alpha 1-PI did not form normal complexes witrh serine proteinases. These findings suggest that the inhalation of ozone could inactivate alpha 1-PI on the airspace side of the lung to create a localized alpha 1-PI deficiency, which might contribute to the development of emphysema.
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PMID:Ozone inactivation of human alpha 1-proteinase inhibitor. 690 14

The amino acid sequence of the sulfate-binding protein from Salmonella typhimurium LT2 was determined by automated sequenator analysis of whole protein and fragments derived by chemical and enzymatic cleavage of whole protein. The fragments were products of limited trypsin digestion at arginine, cleavage at tryptophan by BrNps-skatole and o-iodosobenzoic acid, digestion with the protease from Staphylococcus aureus V8 at Glu-X bonds, cleavage by hydroxylamine at Asn-Gly bonds, and subdigestion with trypsin, chymotrypsin, and the Staphylococcus protease. The COOH-terminal sequence was confirmed using carboxypeptidase B and amino acid analysis. The sulfate-binding protein was determined to contain a single polypeptide of 310 residues with a molecular weight of 34,667 calculated from the sequence.
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PMID:Amino acid sequence of the sulfate-binding protein from Salmonella typhimurium LT2. 698 15

The possibility that the rates of acylation of chymotrypsin by certain highly reactive substrates approach the diffusion-controlled limits was investigated by measuring the values of kcat/Km for three substrates as a function of increasing viscosity with sucrose and ficoll as the viscosogenic reagents. The values of Kcat/Km (pH 8.0, 25 degrees C) representing the acylation rate constants are the following: N-(methoxycarbonyl)-L-tryptophan p-nitrophenyl ester, 3.5 x 10(7) M-1 s-1; N-acetyl-L-tryptophan methyl ester, 8 x 10(5) M-1 s-1; N-acetyl-L-tryptophan p-nitroanilide, 300 M-1 s-1. The rate constants decrease significantly with increasing viscosity for the first compound, decrease slightly for the second, and are insensitive to this perturbation for the third. The p-nitroanilide results taken together with the observation that the high concentrations of sucrose or ficoll used produce insignificant changes in kcat for the ester substrates argue against a general nonspecific perturbation in the enzyme structure effected by these reagents. The values of the association rate constants calculated from these results are 9 x 10(7) and 1 x 10(7) M-1 s-1 for the p-nitrophenyl and methyl esters, respectively. The values of kcat/Km divided by the association rate constants show that the rates of acylation by the p-nitrophenyl ester occur at ca. 40% and by the methyl ester at ca. 10% of the diffusion limits. Possibilities involving reorientation of a nonproductively bound substrate within the ES complex or desolvation of part of the active site of the enzyme are considered to account for the lower association rate constant for the methyl as compared to the p-nitrophenyl ester.
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PMID:Investigation of diffusion-limited rates of chymotrypsin reactions by viscosity variation. 707 86

Proteolipid aproprotein (lipophilin) and DM-20 protein from bovine brain white matter proved to be identical in polyacrylamide gel electrophoresis and automated Edman degradation of the N-terminal end over 20 cycles. Lipophilin can be hydrolysed by trypsin, thermolysis, chymotrypsin and subtilisin. We describe here a new, effective and rapid high-performance liquid chromatographic separation method for hydrophilic polypeptides according to molecular mass on an analytical and preparative scale. Three large and several small peptides have been isolated from the tryptic and thermolysinolytic hydrolysate and purified by combined molecular sieve and high-performance chromatographic separation and purification for automated Edman degradation. 40 amino acid residues of the large tryptic fragment and sequences of 43 and 22 amino acids of two thermolysinolytic fragments have been determined. These three polypeptides are partial structures of the l4 kDa large tryptophan fragment 1 or the cyanogen bromide fragment I (18-19 kDa). Thermolysin also releases a polypeptide from incompletely reductively carboxymethylated lipophilin which is cleaved into the large thermolysin fragment mentioned, 22 residues of which were analysed, and a 14 amino acids long sequence of tryptophan fragment IV, described in the previous paper. Reductively carboxymethylated liprophilin, the lysine side chains of which were blocked with maleic anhydride, can be cleaved at arginine specific sites. Bio-Gel P-150 and high-performance chromatographic purification yielded a polypeptide, which upon performic acid oxidation was split into a 15 kDa and a 7.8 kDa polypeptide. The 15 kDa polypeptide resembles the N-terminal end as proven by 31 cycles in Edman degradation. The 7.8 kDa polypeptide corresponds to the 72 amino acid C-terminal sequence, which equals cyanogen bromide fragments II, III and IV and embraces tryptophan fragment IV.
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PMID:Analysis of the primary structure of the strongly hydrophobic brain myelin proteolipid apoprotein (lipophilin). Isolation and amino acid sequence determination of proteolytic fragments. 714 16

The chemical cleavage of lipophilin (proteolipid apoprotein) from bovine brain white matter with HBr/dimethyl sulfoxide at the tryptophan residues, under conditions adapted to this hydrophobic protein, releases four fragments with approximate molecular masses 14 kDa (Trp I), 6.8 kDa (Trp IV), 5.2 kDa (Trp III) and 2.1 kDa (Trp II). These fragments have been separated and purified by a combination of solvent distribution, molecular sieve chromatography (Bio-Gel P-150) and high-performance liquid chromatography for automated Edman degradation and combined gas-liquid chromatography/mass spectroscopy. The complete amino acid sequences of Trp II and III and large sequences of Trp I are reported in this communication. The amino acid sequence of Trp IV and the sequences of peptides releasable from lipophilin by proteolytic enzymes (trypsin, thermolysin, subtilisin, chymotrypsin) have been described in previous reports from this laboratory. Despite two small gaps in the complete primary structure of lipophilin from myelin of central nervous system, our sequence data suggest the arrangement of four long hydrophobic sequences (30-40 apolar amino acid residues) within the hydrophobic core of the myelin lipid bilayer, linked by three hydrophilic regions at the aqueous membrane interphase. These features lend lipophilin the properties of a polytopic membrane protein.
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PMID:Lipophilin (proteolipid apoprotein) of brain white matter. Purification and amino acid sequence studies of the four tryptophan fragments. 717 28

The chicken hepatic lectin is involved in the clearance of glycoproteins from circulation (Kawasaki, T., and Ashwell, G. (1977) J. Biol. Chem. 252, 6536-6543). The complete amino acid sequence of chicken hepatic lectin has been established by analysis of peptides generated by chemical cleavage at methionine or tryptophan residues. Larger BrCN fragments were further digested with trypsin, chymotrypsin, and clostripain. All sequences were determined by automated sequential Edman degradation. Extensive use was made of high performance liquid chromatography in the purification of peptides and identification of phenylthiohydantoin derivatives of amino acids. The complete sequence is: (formula: see text). The stretch of uncharged amino acids from residue 25 to 48 is a possible membrane-interaction region. Carbohydrate is attached to residue 67.
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PMID:Complete amino acid sequence of a membrane receptor for glycoproteins. Sequence of the chicken hepatic lectin. 724 Jan 75


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