Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: C01B12 .4
 471,332 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinetic specificities of BPN' and Carlsberg subtilisins [EC 3.4.21.14] were examined with various nucleus-substituted derivatives of Nalpha-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of alpha-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two sutilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar trans-p-phenylazobenzoyl group at the OH-function, with the smallest Km value showed that these enzymes possess a more extended aromatic binding site than has so far been demonstrated. Ac-Phe(4-NO2)-OMe was remarkable in being hydrolyzed with a particularly large kcat value (5,500 +/- 700 s-1 at pH 7.8 for Carlsberg subtilisin). Ac-Phe(4-NO2)-OMe and Ac-Tyr-OMe were distinguished by Carlsberg subtilisin in terms of kcat but not by BPN' subtilisin, suggesting that the specificity site of the former is more sensitive to a small change in size of substituent than that of the latter. Ac-Trp(NCps)-OMe and Ac-Trp(NCps)-OH were bound to the enzyme's active site but in a competitive manner. A difference in the standard free energies of binding between the two enzymes may indicate that the hydrophobic cleft of Carlsberg subtilisin is somewhat deeper and/or narrower than that of BPN' subtilisin.
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PMID:Kinetic specificities of BPN' and Carlsberg subtilisins. Mapping the aromatic binding site. 10 40

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.
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PMID:Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin. 13 79

The inhibition of plasmin, (EC 3.4.21.7), thrombin (EC 3.4.21.5), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) by antiplasmin, the recently described fast-reacting plasmin inhibitor of human plasma, was studied. To determine the quantitative importance of antiplasmin relative to the other plasma protease inhibitors, enzyme inhibition assays were performed on whole plasma and on plasma specifically depleted in antiplasmin, after addition of excess enzyme. Plasmin was the only enzyme for which the inhibitory capacity of antiplasmin-depleted plasma was lower than that of normal plasma. To determine the affinity of the enzymes for antiplasmin, as compared to the other inhibitors, various amounts of enzymes were added to normal plasma and the formation of enzyme-antiplasmin complexes studied by crossed immunoelectrophoresis using specific antisera against antiplasmin. Plasmin and trypsin, but not thrombin or chymotrypsin formed complexes with antiplasmin. It is concluded that antiplasmin is the only fast-reacting plasmin inhibitor of human plasma. It is also a fast-reacting inhibitor of trypsin but only accounts for a very small part of the fast-reacting trypsin-inhibitory activity of plasma. This can be explained by the low concentration of antiplasmin (1 muM) in normal plasma, compared to the other inhibitors (e.g. alpha1-antitrypsin: 40-80 muM).
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PMID:The interaction in human plasma of antiplasmin, the fast-reacting plasmin inhibitor, with plasmin, thrombin, trypsin and chymotrypsin. 14 66

In order to clarify the function of the carbohydrate moiety of bovine kappa-casein, kappa-casein components having different carbohydrate contents were prepared by DEAE-cellulose chromatography. Five adsorbed fractions so obtained had an identical peptide chain and contained carbohydrate moieties of increasing size in the order of components P-2, P-3, P-4, P-5 and P-6. The subsceptibility of kappa-casein components, having different carbohydrate contents, to various proteases was examined. kappa-Casein components were subjected to calf rennin [chymosin; EC 3.4.23.4], bovine trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1], pronase [EC 3.4.24.4] and human plasmin [EC 3.4.21.7]. The component containing a larger carbohydrate moiety was less susceptible to hydrolysis than the component containing a smaller carbohydrate moiety. Rennin, trypsin, alpha-chymotrypsin and pronase hydrolyzed each component with a different reaction rate. On the contrary, human plasmin hydrolyzed component P-2, but did not hydrolyze component P-5. These results indicate that the carbohydrate moiety of kappa-casein components to various proteases.
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PMID:Susceptibility of kappa-casein components to various proteases. 15 50

We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2-24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or alpha-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16-48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10(6) cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, alpha(1)-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.
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PMID:Proteases induce secretion of collagenase and plasminogen activator by fibroblasts. 20 72

Isolated pancreatic acini were prepared by a new method from mouse and rat pancreases by digestion with purified collagenase and chymotrypsin followed by mechanical shearing. Acini were structurally similar to those of the intact pancreas, having a normal luminal structure but with the basal acinar cell membranes exposed to the incubation medium. Amylase release in response to both cholinergic analogues and the cholecystokinin analogues caerulein and pentagastrin was comparable to that of the intact pancreas, but was much greater than previously reported for isolated acinar cells. Cholinergic-stimulated release was inhibited by atropine with a Ki value of 1.4 nM which is comparable to other muscarinic receptors. All agonists tested, when added at supramaximal concentrations, produced a submaximal release of amylase even though ATP levels and the release of slowly exchanging 45Ca2+ were normal or increased. Acini releasing amylase submaximally after being exposed to supramaximal concentrations of carbachol failed to respond to a maximal amount of caerulein or to the Ca2+ ionophore A23187. It is concluded that the decreased response (desensitization) is a postreceptor phenomenon and possibly mediated by Ca2+ itself.
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PMID:Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini. 21 42

Biphasic kinetic data were obtained when trypsin (EC 3.4.21.4) which had previously been complexed with a thiol-containing inhibitor (present in Ehrlich ascites tumour cells) was incubated with incremental additions of periodate. At low concentrations of periodate the trypsin was re-activated whilst at higher concentrations of periodate the trypsin was irreversibly inhibited. This biphasic reactivation followed by inhibition was also demonstrated when trypsin was first inhibited by dithiothreitol and followed by incremental addition of periodate. Similar results were obtained with chymotrypsin (EC 3.4.21.1). Incremental additions of either dithiothreitol or periodate caused inhibition of both these enzymes. The biphasic kinetic data can be explained in terms of reduction and oxidation of a significant disulphide bond in both trypsin and chymotrypsin which can be cleaved by thiols in a disulphide exchange reaction [1]. This bond is thought to maintain the active centres of each of these enzymes in a conformation sterically favourable for enzymic cleavage of specific peptide bonds in the protein substrates (polymeric collagen fibrils and casein) employed in this study.
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PMID:Inhibition of trypsin and chymotrypsin by thiols. Biphasic kinetics of reactivation and inhibition induced by sodium periodate addition. 22 48

Three cationic proteins from the granules of human neutrophil granulocytes were obtained in a high degree of purity be means of affinity chromatography on 4-phenylbutylamine-Sepharose. Together with lysozyme, the three cationic proteins exhibit the highest electrophoretic mobility toward the cathode in acrylamide gels at moderately acid pH, among the granule constituents that are solubilized in 0.1 M phosphate buffer, pH 7.0, containing 1 M NaCl. The three cationic proteins represent a group of "neutral proteases" distinct from elastase and collagenase. They hydrolyze casein, azocasein and the chymotrypsin substrate N-acetyl-L-tyrosine ethyl ester. Optimal activity is found at pH 7.4-7;5. The enzymes are inhibited by the specific chymotrypsin inhibitor N-tosyl-L-phenylalanylchloromethane and by the naturally occurring inhibitors alpha-antichymotrypsin, alpha-1-antitrypsin, as well as by the trypsin inhibitors from soy beans and limabeans.
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PMID:Cationic proteins from human neutrophil granulocytes. Evidence for their chymotrypsin-like properties. 23 18

Elastolytic enzyme was purified and crystallized from culture fluid of Flavobacterium immotum No. 9-35. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight was determined by Sephadex G-100 gel filtration to be 13,000. The isoelectric point was between pH 8.3 and 8.9. The optimum pH of the enzyme was 7.2 for elastolytic activity. The purified enzyme showed not only elastolytic activity, but also non-specific proteolytic activity against various other proteins. Milk-clotting activity was also observed. The enzyme did not act on keratin, collagen, or fourteen amino acid esters, including N-benzoyl-L-alanine methyl ester, N-benzoyl-L-arginine ethyl ester, and N-acetyl-L-tyrosine ethyl ester, which were typical substrates of pancreatic elastase [EC 3.4.21.11], trypsin [EC 3.4.21.4], and chymotrypsin [EC 3.4.21.1], respectively. However, the enzyme selectively hydrolyzed elastin when both elastin and albumin were present in the reaction mixture. The enzyme was inhibited by o-phenanthroline and various heavy metals such as cadmium, lead, zinc, and mercury. Various inhibitors, such as diisopropyl phosphofluoridate, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, trypsin inhibitor, iodoacetamide, etc., had no effect on the elastolytic activity.
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PMID:Purification and properties of elastolytic enzyme from Flavobacterium immotum. 23 95

Kinetic parameters for carboxypeptidase Y [EC 3.4.12.1], characterized as a nonspecific enzyme, are given for the hydrolysis of a series of acylated peptides, acylated amino acid esters, and amides. We confirmed that the enzyme released COOH-terminal proline and beta-alanine at an appreciable rate, as well as neutral amino acids with aromatic and aliphatic side chains at a very high speed. The rates of hydrolysis of ester and amide substrates were compatible with those produced by chymotrypsin [EC 3.4.21.1]. Stereospecificity was also demonstrated by the failure to hydrolyze peptide, ester, amide, and anilide substrates containing a D-amino acid. The effects of pH, solvents, and salt concentrations on the kinetic parameters of hydrolysis of peptide and ester substrates are also described.
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PMID:Kinetic studies of carboxypeptidase Y. I. Kinetic parameters for the hydrolysis of synthetic substrates. 23 4


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