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)

Antihemorrhagic proteins from Crotalus atrox serum were tested for their ability to inhibit the proteolytic activity of the hemorrhagic toxin-e from Crotalus atrox venom and of several other proteolytic enzymes: trypsin, collagenase and thermolysin. The antihemorrhagic proteins inhibited the proteolytic activity of hemorrhagin-e when tested on gelatin type I and collagen type IV, the proteolytic activity of trypsin on photofilm gelatin and the proteolytic activity of whole venom when tested on azocollagen and photofilm gelatin. The antihemorrhagins failed to inhibit the proteolytic activity of trypsin when tested on the specific synthetic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester (APNE), the activity of microbial collagenase on N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) or on azocollagen and the activity of thermolysin on N-(3-[2-furyl]acryloyl)-Gly-Leu amide (FAGLA). It is tentatively suggested that the antihemorrhagins from snake blood serum are proteinase inhibitors that underwent specialization towards the neutralization of the proteolytic activity of hemorrhagic toxins.
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PMID:Inhibition of the proteolytic activity of hemorrhagin-e from Crotalus atrox venom by antihemorrhagins from homologous serum. 151 50

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

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well.
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PMID:Limited proteolysis as a probe of conformational changes in aspartate aminotransferase from Sulfolobus solfataricus. 155 94

Neutral proteolytic activity, having a pH optimum of about 7, was present in the high molecular weight fraction of bovine interphotoreceptor matrix (IPM) separated by gel filtration on Sephacryl S-500. The enzyme(s) was active toward a number of exogenous substrates, including albumin, Azocoll, and gelatin. However, it was inactive toward a synthetic substrate for bacterial collagenase. Proteolytic activity was proportional to protein; however, the time course of the reaction was nonlinear, suggesting that "activation" of a precursor form might be necessary. Of a number of specific inhibitors tested, those directed toward metalloproteinases (1,10-phenanthroline greater than EDTA greater than EGTA) proved most effective. While activity was also inhibited by sulfhydryl reagents and dithiothreitol, inhibitors specific for cysteine proteinases were ineffective. Higher specific activity was present in IPM obtained from retinal pigment epithelium (RPE) than from retina. An endogenous proteinase inhibitor(s) was also found in IPM from both RPE and retina. It was effective against the endogenous metalloproteolytic activity of IPM and also against thermolysin, but not against trypsin or papain. Fractionation of IPM on Sephacryl S-500 revealed a broad peak of inhibitory activity at molecular weights of less than 10(5) daltons. This is the first report of the presence of neutral proteolytic activity and metalloproteinase inhibitor(s) in bovine IPM. These materials may function in concert to maintain the proper level of various components within this matrix.
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PMID:The presence of neutral metalloproteolytic activity and metalloproteinase inhibitors in the interphotoreceptor matrix. 155 92

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 accessible surface, described by Lee and Richards (the L&R surface: J. Mol. Biol. 1971, 55, 379), has remarkably useful properties for displaying ligand-protein interactions. The surface is placed one van der Waals radius plus one probe radius away from the protein atoms. The ligands are displayed in skeletal form. With a suitable probe radius, those parts of the ligand in good van der Waals contact with the protein binding site are found superimposed on the L&R surface. Display of the surface using parallel contours therefore provides a very powerful guide for interactive drug design because only ligand atoms lying on or close to the surface are in low-energy contact. The ability of the surface to accurately display steric complementarity between ligands and proteins was optimized using data from small molecule crystal structures. The possibility of displaying the chemical specificity of the binding site was also investigated. The surface can be colored to give precise information about chemical specificity. Electrostatic potential, electrostatic gradient, and distance to hydrogen-bonding groups were tested as methods of displaying chemical specificity. The ability of these methods to describe the complementarity actually observed in the interior of proteins was compared. High-resolution crystal data for ribonuclease and trypsin was used. The environment surrounding extended peptide chains in the protein was treated as a virtual binding site. The peptide chain served as a virtual ligand. This large sample of experimental data was used to measure the correlation between type of ligand atom and the calculated property of the nearest binding site surface. The best correlation was obtained using hydrogen-bonding properties of the binding site. Using this parameter the surface could be divided into three separate zones representing the hydrophobic, hydrogen-bond-acceptor, and hydrogen-bond-donor properties of the binding site. The percentage of hydrophobic ligand atoms found to lie closest to the hydrophobic protein surface was 91%. The equivalent scores for ligand hydrogen-acceptor atoms and hydrogen-donor atoms found at the corresponding complementarity zone were 94% and 91%. The surface zones can be readily displayed using three colors. To test the method on real ligand/binding site interactions, nine thermolysin-inhibitor complexes of known structure were evaluated using the parameters and criteria derived from the protein-packing study and a correlation between complementary contacts and logarithm of potency was obtained which had an r2 of 0.99.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Definition and display of steric, hydrophobic, and hydrogen-bonding properties of ligand binding sites in proteins using Lee and Richards accessible surface: validation of a high-resolution graphical tool for drug design. 158 50

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

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.
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PMID:Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies. 161 9

The photoaffinity reagent 8-[(4-azidophenyl)-methoxy]-1-tritiomethyl-2, 3-dimethylimidazo-[1,2-alpha]pyridinium iodide ([3H]mDAZIP) has been synthesized and used to photoinactivate and label purified hog gastric H+,K(+)-ATPase. The specific (K(+)-sensitive) components of both photoinactivation and labeling showed dependences on inhibitor concentration consistent with covalent modification at an extracytoplasmic site of reversible K(+)-competitive binding in the dark. The maximum amount of specific labeling (1.2 nmol/mg) was similar to the number of phosphorylation sites measured (1.0 +/- 0.14 nmol/mg). Specific labeling was distributed 76% on the alpha chain, 18% on the beta chain, and 6% on undefined peptides. Various digestions with trypsin, protease V8, and thermolysin were employed to fragment the labeled enzyme. Gasphase sequencing of the radioactive peptides identified the major site of specific labeling to be within a region where only two stretches of amino acids (Leu105 to Ile126 and Leu139 to Phe155, designated H1 and H2, respectively) are predicted to span the membrane. This in turn suggested that the labeling site was located within or close to the proposed loop between them (Gln127 to Asn138). A computer-driven energy minimization protocol yielded a loop structure to which SCH 28080 (the parent structure of [3H]mDAZIP) could be docked. Conversely, modeling of the corresponding region of Na+,K(+)-ATPase (a homologous enzyme with much lower affinity for SCH 28080) yielded no apparent binding site. Similarities in the inhibition of H+,K(+)-ATPase by SCH 28080 and of Na+,K(+)-ATPase by ouabain lead to the hypothesis that, in each case, inhibitor binding to E2-P is associated with an increase in the hydrophobicity of the environment of the loop between H1 and H2.
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PMID:Identification of an extracytoplasmic region of H+,K(+)-ATPase labeled by a K(+)-competitive photoaffinity inhibitor. 165 68


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