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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine and human thyroglobulin show two closely migrating bands in reducing SDS-PAGE. Limited digestion with chymotrypsin, trypsin and thermolysin converted the slower band of the doublet into a peptide identical to the faster band, with an apparent mass of 270 kDa, in both species. The starting point of the faster band of the doublet was established at Ileu 520 with native bovine Tg and at Ser 503 with native human Tg, and at Ser 503 and Ser 504 with chymotrypsin-digested bovine and human Tg, respectively. These data explain the electrophoretic heterogeneity of thyroglobulin and unveil a region highly susceptible to proteolysis at about 500 residues from the NH2-terminus of the molecule.
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PMID:The origin of the electrophoretic doublet of thyroglobulin. 151 Jun 54

The cell-extracellular matrix junction, which includes the cell wall and the outer surface of the plasma membrane, may be an essential region for the perception of gravity by the internodal cells of Chara corallina. Typically, when an internodal cell is oriented vertically, the downwardly directed cytoplasmic stream travels at a velocity that is 10% faster than that of the upwardly directed stream. However when the cells are treated with impermeant hydrolytic enzymes that partially digest cellulose or hemicellulose, the cells lose their ability to respond to gravity even though streaming continues. By contrast, enzymes that digest pectins have no effect on the gravity-induced polarity of cytoplasmic streaming. Furthermore, gravisensing is sensitive to protease treatment; Proteinase K, thermolysin and collagenase but not trypsin, alpha-chymotrypsin or carboxypeptidase B, inhibit gravisensing. These findings indicate that proteins in the cell-extracellular matrix junction may be required for gravisensing. Moreover, the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibits gravisensing in a concentration-dependent manner, indicating that the gravireceptor may be an integrin-like protein. The macromolecules necessary for gravisensing have been localized to the cell ends. As a consequence of the exoplasmic site of action of the enzymes and the tetrapeptides, we interpret the results to mean that they are acting on the gravireceptor, although we cannot eliminate the possibility that they are acting on the signal transduction chain. On the whole, our observations indicate that the cell-extracellular matrix junction is a sine qua non for graviperception in statolith-free Chara internodal cells and we suggest that the gravireceptor is located in this region.
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PMID:The contribution of the extracellular matrix to gravisensing in characean cells. 152 45

Three-headed Tetrahymena 22S ciliary dynein was found to consist of three heavy chains (HCs) and decompose into two-headed and single-headed fragments upon chymotrypsin digestion. The three HCs (A alpha, A beta, and A gamma) were immunologically different, and presumed to be located on each of the head regions. The two-headed fragment contained A beta and A gamma HCs, while the A alpha HC originated in the single-headed fragment. Both fragments were associated with ATPase activity (Toyoshima, Y. (1987a) J. Cell Biol. 105, 887-895 and Toyoshima, Y. (1987b) J. Cell Biol. 105, 897-901). Using the two-headed dynein fragment, we attempted to determine the site of ATP hydrolysis in the fragment. After digestion of the fragment with 100 micrograms/ml thermolysin for 45 min, we noted eight thermolysin-digested polypeptides (TH 1, 2, 3, 4, 5 alpha, 5 beta, 6 alpha, and 6 beta). By precisely analyzing the degradation process and the products using peptide mapping, immunoblotting and high pressure liquid chromatography, it appeared that the two-headed fragment is dissociated as two separate fragments, each of which contained A beta or A gamma HC. Thermolysin digests, TH 1, 2, 5 alpha and 6 beta were found to be derived from A beta HC, while TH 3, 4, 5 beta and 6 alpha originated in the A gamma HC. Based on the measurements of ATPase activity of these polypeptides, we concluded that the ATPase site is located in the A beta and A gamma HCs, which may have their origins in each head of the two-headed fragment of Tetrahymena 22S ciliary dynein.
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PMID:ATPase sites in two-headed fragment of Tetrahymena 22S ciliary dynein. 153 22

Bovine liver glutamate dehydrogenase reacts with the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine (5'-FSBAzA) in a two-step process: a dark reaction yielding about 0.5 mol of -SBAzA/mol of subunit by reaction through the fluorosulfonyl moiety, followed by photoactivation of the azido group whereby covalently bound -SBAzA becomes cross-linked to the enzyme [Dombrowski, K. E., & Colman, R. F. (1989) Arch. Biochem. Biophys. 275, 302-308]. We now report that the rate constant for the dark reaction is not reduced by ADP or GTP, but it is decreased 7-fold by 2 mM NADH and 40-fold by 2 mM NADH + 0.2 mM GTP, suggesting that 5'-FSBAzA reacts at the GTP-dependent NADH inhibitory site. The amino acid residues modified in each phase of the reaction have been identified. Modified enzyme was isolated after each reaction phase, carboxymethylated, and digested with trypsin, chymotrypsin, or thermolysin. The digests were fractionated by chromatography on a phenylboronate agarose column followed by HPLC. Gas-phase sequencing of the labeled peptides identified Tyr190 as the major amino acid which reacts with the fluorosulfonyl group; Lys143 was also modified but to a lesser extent. The predominant cross-link formed during photolysis is between modified Tyr190 and the peptide Leu475-Asp476-Leu477-Arg478, which is located near the C-terminus of the enzyme. Thus, 5'-FSBAzA is effective in identifying critical residues distant in the linear sequence, but close within the regulatory nucleotide site of glutamate dehydrogenase.
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PMID:Identification of amino acids modified by the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine in the reduced coenzyme regulatory site of bovine liver glutamate dehydrogenase. 156 33

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. 156 52

The differential cleavage of surface proteins of Borrelia burgdorferi IRS strains by several proteases was examined. Proteinase K, trypsin, chymotrypsin and thermolysin all cleaved the outer surface protein B (OspB) to undetectable levels by Coomassie Brilliant Blue staining, whereas some residual protein was detected by immunoblotting with polyclonal and monoclonal antibodies. Not even antigenic fragments were detectable by immunoblotting with 1A8 monoclonal antibody reactive with OspB. Less effective or ineffective was the cleavage of OspB by V8 protease and proteinase A, respectively. The outer surface protein A was cleaved only by proteinase K. The effect of trypsin on borreliae viability and adhesion to cultured cells was also studied. The trypsin treatment of borreliae did not impair the viability of organisms which continued to synthesize the cleaved OspB. The attachment of B. burgdorferi to HEp-2 cells was reduced by 41% after treatment with trypsin, whereas preincubation of borreliae with monoclonal antibody 1A8 and guinea pig immune serum reduced the adhesion of borreliae to the cells by 32% and 87%, respectively.
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PMID:Differential cleavage of surface proteins of Borrelia burgdorferi by proteases. 160 94

It was found that the fluorescence of 9,10-dioxa-syn-3,4,6,7-tetramethylbimane (bimane) can be quenched in the presence of dimethylaminoazobenzensulfonyl (Dabsyl) group. New combination of bimane (fluorophor) and dabsyl group (quencher) was applied to the syntheses of intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. Bimane peptides containing dabsyl group were prepared, and were shown to be useful fluorogenic substrates for the assay of endopeptidases such as chymotrypsin, collagenase and thermolysin.
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PMID:Fluorogenic bimane substrates with dabsyl group for endopeptidases; chymotrypsin, collagenase and thermolysin. 166 27

Covalent binding of proteinases by human alpha 2-macroglobulin (alpha 2M) results primarily from the formation of stable epsilon-Lys-gamma-Glu isopeptide bonds. Cross-linking engages 12, 13, and 10 of the 14, 14, and 11 Lys residues in chymotrypsin, trypsin, and subtilisin, respectively, and reaction with the alpha-amino group of the C-chain of chymotrypsin and the B-chain of beta-trypsin is also seen. In contrast, cross-linking engages only 6 of the 11 Lys residues in thermolysin. In each of these proteinases, a few residues react to the greatest extent: Lys36, Lys79, Lys87, and Lys93 in chymotrypsin; Lys87, Lys109, Lys222, and Lys239 in trypsin; Lys12, Lys43, and Lys141 in subtilisin; and Lys210 and Lys219 in thermolysin. In elastase, 1 of the 3 Lys residues (Lys87) is tentatively identified as being cross-linked. Formation of unstable bonds judged to be mainly p-tyrosyl-gamma-glutamyl esters can also be significant for some proteinases. In each of the proteinases, several of the strongly reacting Lys residues are located relatively close to each other, presumably reflecting steric constraints within the alpha 2M-proteinase complexes as they form. Proteinases are covalently bound to alpha 2M to one or two of its COOH-terminal bait region-cleaved half-subunits. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of the high molecular weight cross-linked species indicates that binding of a proteinase through two cross-links occurs not only within the 360-kDa disulfide-bridged alpha 2M dimer but also between the two dimers in the alpha 2M tetramer.
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PMID:Localization of epsilon-lysyl-gamma-glutamyl cross-links in five human alpha 2-macroglobulin-proteinase complexes. Nature of the high molecular weight cross-linked products. 169 79

Rat alpha 1-inhibitor-3 is a 180-kDa monomeric proteinase inhibitor found in high concentration in rat plasma. By several criteria it has been shown to be a member of the family of alpha-macroglobulin proteinase inhibitors often exemplified by the tetrameric human alpha 2-macroglobulin. We have used limited proteolysis of rat alpha 1-inhibitor-3 to probe the domain structure of this family of proteins. Proteinases of different specificities, including trypsin, chymotrypsin, thermolysin, and Staphylococcus aureus V8 proteinase, were employed and a common fragmentation pattern was observed when the reaction products were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These fragments were electrotransferred to polyvinylidene difluoride membranes and subjected to NH2-terminal amino acid sequence analysis in order to position them within the context of the primary structure. The fragmentation pattern may define the domain structure of alpha 1-inhibitor-3 and serve as a model for the domain organization of the family of alpha-macroglobulin proteinase inhibitors.
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PMID:Limited proteolysis of the alpha-macroglobulin rat alpha 1-inhibitor-3. Implications for a domain structure. 171 Feb 22

The presence of a low molecular mass polypeptidic antigen in Borrelia burgdorferi was described. The protein was exposed at the bacterial surface since it was clearly identified by mAb 3H4 using the immunofluorescence test performed with living bacteria. This antigen was cleaved by proteinase K treatment, whereas it was resistant to the action of chymotrypsin, trypsin and thermolysin. Western blotting analysis of the immunological reactivity of this antigenic structure performed using monoclonal antibody, mouse-immune ascitic fluids raised against B. burgdorferi and other spirochetes, sera from patients with Lyme disease and other infirmities in which false positive results in serological tests for B. burgdorferi have been described, demonstrated that this protein expresses only species-specific epitopes which may be recognized during human B. burgdorferi infections.
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PMID:Immunological characterization of a low molecular mass polypeptidic antigen of Borrelia burgdorferi. 172 56


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