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)

The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.
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PMID:Reinvestigation of the phenacyl bromide modification of alpha-chymotrypsin. 0 93

A reinvestigation of the modification reactions of alpha-chymotrypsin with phenacyl bromide was carried out. Results conclusively demonstrate that the chemically and physically different modified enzymes prepared at pH 4 and at pH 7 both contain the phenacyl group at methionine-192 in the sulphonium salt form. Evidence to suppoort this conclusion derives from 13C nuclear-magnetic-resonance spectroscopic observations on [methylene-13C]phenacyl-enriched enzymes. More conclusively, the methionine-192-containing C-chain, derived by performic acid oxidative cleavage of radioactively-labelled enzyme prepared at pH 7, was shown to contain the phenacyl moiety and to undergo dealkylation by 2-mercaptoethanol with loss of this moiety. In addition, thermolytic cleavage of the high-pH enzyme results in fragmentation of the polypeptide chain in a fashion analogous to model reactions of phenacylmethionyl dipeptides and other methionine-192 sulphonium salts. A rationalization of the unusual nature of the high-pH phenacyl-modified enzyme based on the irreversible formation of stable conformation in which the phenacyl moiety is rigidly located in interior regions of the enzyme is presented and discussed.
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PMID:Evidence for a pH-dependent irreversible formation of a stable conformation of phenacyl-alpha-chymotrypsin. 2 56

Microorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3.4.11.8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the enzyme. The enzyme was purified from lysozyme-lysate of the bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogenous. The enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the enzyme. The molecular weight was estimated to be 72,000 by the gel filtration method and to be 24,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a subunit oligomer, presumably trimer. The enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, beta-naphthylamine, alpha-naphthol, and 4-methylumbelliferone, but was completely inert towards various peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and alpha-chymotrypsin.
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PMID:Purification and characterization of L-pyrrolidonecarboxylate peptidase from Bacillus amyloiliquefaciens. 2 93

A cationic polypeptide growth factor, isolated from human serum and purified to homogeneity, has stimulated the replication of density-inhibited BALB/c 3T3 cells. It has a molecular weight of 1.3 x 10(4) daltons and an isoelectric point of 9.7. Trypsin or chymotrypsin digestion reduces the growth-stimulatory activity by 75%, whereas 2-mercaptoehanol completely abolishes it. The growth factor is heat-stable (100 degrees C X 10 min) and free of insulin-like activity. The highly purified serum growth factor has been labeled with 125I, and an antiserum to the growth factor was produced in the rabbit. A specific, highly sensitive radioimmunoassay has been developed. Factors with growth-stimulating activity have also been detected in human platelets and human pituitary gland extracts. Platelets and pituitary glands have antigenic determinants that are recognized by antibodies to the serum growth factor. The platelet and pituitary gland growth factors are also cationic and heat stable, and are destroyed by 2-mercaptoethanol. Thus the human serum, platelet, and pituitary gland growth factors have similar properties.
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PMID:Growth factors derived from human serum, platelets, and pituitary: properties and immunologic cross-reactivity. 8 59

The Rho(D) antigen of red cell membranes was solubilized using ethylene-diamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, and pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rho(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rho(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rho(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rho(D) molecule in its natural environment. The solubilized Rho(D) molecules are apparently not dependent on lipid for their Rho(D) activity.
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PMID:Studies on the characterization of the Rho(D) antigen. 10 79

Limited proteolysis of bovine colostral IgG1 by trypsin caused loss of specific antibody activity but column chromatography showed that relatively little cleavage into fragements had occurred. Polyacrlamide-agarose SDS electrophoresis of the 2-mercaptoethanol-treated digest revealed, however, that extensive cleavage of light chains had occurred even though most of the material before reduction had a mol. wt close to that of undigested IgG1. Although a Fab-type fragment was detected in the digest by immunoelectrophoresis it appeared to be only a minor component. Chymotrypsin had little effect upon either the structure or antibody activity of IgG1. These findings may explain the effect of trypsin and chymotrypsin on the bactericidal activity of colostral antibodies.
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PMID:The effect of limited proteolysis by trypsin and chymotrypsin on bovine colostral IgG1. 32 43

Cytosol of horse blood polymorphonuclear leucocytes contains an inhibitor active against neutral proteinases from the granules of these cells and against chymotrypsin, elastases I and II from pig pancreas, but not against trypsin. A method has been elaborated to isolate and purify this inhibitor by means of salting out with ammonium sulphate (45---70% saturation), followed by chromatography and rechromatography on a column of DEAE-Sephadex A-50. The preparation obtained is homogeneous during polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecylsulphate after reduction with 2-mercaptoethanol. It is a thermolabile protein soluble at its isoelectric point (pH 5.38) and having a molecular weight of 35200 but aggregating in solutions of low ionic strength and irreversibly precipitating during dialysis at pH 4.0. The results obtained show the similarity of this inhibitor isolated from horse blood leucocyte cytosol to other inhibitors isolated from human or pig blood leucocytes.
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PMID:A polyvalent proteinase inhibitor from horse-blood-leucocyte cytosol. Isolation, purification and some molecular parameters. 84 42

The recombinant gene for hepatitis B core antigen (HBcAg) was cloned and expressed, and the protein was purified from Escherichia coli cultures. Purified HBcAg was tested for the effects of various physical and chemical agents on its immunoreactivity by a paramagnetic particle-based enzyme immunoassay. Recombinant HBcAg retained its immunoreactivity when heated at 70 degrees C for 60 min but was inactivated at 85 degrees C in 10 min. It was stable between pHs 5 and 10.5 but not at pHs 2 and 13.5. Treatment with sodium dodecyl sulfate (SDS), ethanol, and methanol caused a significant loss in HBcAg reactivity. The proteolytic enzymes papain and bacterial protease (type VIII from Bacillus licheniformis) degraded HBcAg significantly, but trypsin and chymotrypsin did not. The effect of combined SDS and 2-mercaptoethanol on recombinant HBcAg was an immediate loss in immunoreactivity, followed by rapid recovery to about 50% of the initial level. This level was maintained for 24 to 48 h and was followed by an almost total loss of HBcAg in about 120 h.
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PMID:Stability of the recombinant hepatitis B core antigen. 162 88

Purified Escherichia coli Shiga-like toxin II variant (SLT-IIv) was characterized with regard to selected physical, chemical, and biological properties. N-terminal amino acid sequencing confirmed the identities of 33,000-, 27,500-, and 7,500-molecular-weight (MW) bands seen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified SLT-IIv as the A subunit, A1 fragment, and B subunit, respectively. The arginine-serine bond between amino acids 247 and 248 in the A subunit was determined to be the site for proteolytic cleavage into A1 and A2 fragments. As with other SLTs, gel filtration chromatography of SLT-IIv gave estimates of the MW of holotoxin that were variable and less than predicted for a 1-A-subunit-5-B-subunit configuration. The MWs were estimated to be 40,000 and 43,000 by Sephacryl S-100 and Sephadex G-100 and less than 2,000 by Bio-Sil Sec-250 gel filtration chromatography. The isoelectric point of SLT-IIv holotoxin was 9.0. Cytotoxicity of SLT-IIv was destroyed by heating at 65 degrees C for 30 min and by incubation with 2-mercaptoethanol and dithiothreitol, but it increased 30-fold by incubation with trypsin, chymotrypsin, or pepsin and 2-fold by incubation with thermolysin. SLT-IIv cytotoxic activity was stable at neutral and alkaline pH values but was lost at pHs 3, 4, and 5. SLT-IIv was stable in fluid from the anterior and posterior small intestines of pigs but was not enterotoxic in pig intestinal loops. The smallest doses of SLT-IIv that inhibited protein synthesis in porcine endothelial cells and Vero cells were 0.1 ng and 0.1 fg, respectively.
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PMID:Physicochemical and biological properties of purified Escherichia coli Shiga-like toxin II variant. 200 12

Monoclonal antibodies (MAbs) are useful reagents for the study of the structure and evolution of specific epitopes. Two MAbs of IgG1 isotype, Tf-1 and Tf-2, which bind human transferrin have been produced and characterized. Both specifically recognize transferrin on immunoblots of serum. Proteolytic digestion with papain or chymotrypsin destroys the epitope recognized by Tf-1 but not Tf-2, demonstrating that the MAbs recognize distinct epitopes. Both epitopes are not recognized after treatment with 2-mercaptoethanol, suggesting that disulfide bond dependent tertiary structure is necessary for epitope integrity. Removal of carbohydrate moieties by treatment with trifluoromethane sulfonic acid likewise results in loss of reactivity. Neither MAb reacts with transferrin of mouse, rabbit or bovine origin. Both were tested for reactivity to a total of ten primate transferrins and showed different patterns of reaction. Tf-2 recognized human, chimpanzee and gorilla transferrins, whereas Tf-1 reacted with all Old World monkeys and one of three New World monkeys tested. Thus, Tf-1 and Tf-2 recognize transferrin epitopes with differential phylogenetic conservation and which are dependent not only on primary aminoacid sequence, but also upon tertiary structure and glycosylation.
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PMID:Monoclonal antibodies to human transferrin: epitopic and phylogenetic analysis. 245 50


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