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)

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 macromolecule which binds intrinsic factor saturated with vitamin B12 has been solubilized from the guinea-pig ileum by homogenization followed by mechanical disruption without organic solvents or detergents. This intrinsic factor 'receptor' was further purified by precipitation with 30% saturated ammonium sulphate, centrifugation at 105000 g, and filtration through Sephadex G-200. Failure to precipitate the receptor following centrifugation at 105000 g for 3 h and filtration of the receptor with the included volumes through Sepharose 4B and 6B was evidence that it was solubilized. The purification of the receptor was monitored by a radiometric assay where the intrinsic factor-[57Co]vitamin-B12 complex coupled to the solubilized receptor precipitated at 15% sodium sulphate while intrinsic factor-[57Co]B12 alone remained soluble at this salt concentration. This radioassay also permitted the in vitro study of the interaction of the solubilized receptor and intrinsic factor saturated with [57Co]B12. The receptor did not bind intrinsic factor-[57Co]B12 below pH 5 while binding was observed to pH 9.0. Binding was equivalent at 37 degrees C and 25 degrees C, but was markedly reduced at 4 degrees C and 56 degrees C and was destroyed at 100 degrees C. The receptor resisted 60 min of digestion by trypsin, chymotrypsin, pronase and subtilisin. After 180 min digestion, pronase and subtilisin inactivated 90% and 41% of the receptor respectively, whereas trypsin and chymotrypsin inactivated only 21% and 23%. Trisodium EDTA inhibited the binding of intrinsic factor-[57Co]B12 to the receptor and this inhibition could be reversed by the addition of excess Ca2+. Mg2+ and Mn2+ were less effective than Ca2+ for the activity of the receptor. Kinetic analysis of the reaction indicated a maximum velocity of 0.083 nmole IF bound B12/min with a Km of 1.36 x 10(-10) M. The solubilized receptor had a greater affinity for intrinsic factor bound to vitamin B12 than for intrinsic factor free of vitamin B12. The solubilization of this intrinsic factor receptor without chemicals suggests that it is not an integral component of the microvillus membranes hydrophobically bonded to the lipid matrix, but rather a peripheral protein weakly associated with the membrane by non-covalent interaction.
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PMID:Solubilization, partial purification and radioassay for the intrinsic factor receptor from the ileal mucosa. 1 Sep 57

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

It is well known that alpha-chymotrypsin can exist in two major conformational states, only one of which is active. We have examined the pH (pH 2.0--11.0) and salt (ionic strength 0.01--1.0) dependence of the transition between the active and inactive forms in detail. At low pH (pH 2.0--6.0) the equilibrium is very dependent on salt concentration, with high salt concentrations effectively stabilizing the active conformation. This apparent stabilization is an artifact due to the salt-dependent dimerization of alpha-chymotrypsin, and our data show that only active species form dimers and higher aggregates. At neutral pH (6.0--8.0) dimerization is absent, yet an ionic strength dependence remains. The effects show no lyotropic order and appear to be due to preferential salt binding to the active conformation at one or possibly a few sites. Above pH 6 (pH 6.0--11.0), the pH dependence can be described by a two-ionization mechanism at all ionic strengths. We report values for all seven equilibrium constants in the proposed mechanism at four ionic strengths (mu = 0.01, 0.05, 0.2, and 1.0). The transition is the first "refolding" transition to be studied at high precision, but, even so, certain decisions about the mechanism must await higher experimental precision not available with present methods.
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PMID:Refolding transition of alpha-chymotrypsin: pH and salt dependence. 2 61

Proteases capable of activating procollagenase from gingiva and from fibroblast and macrophage monolayer cultures were harvested from homogenates of canine tumor mast cells. The mast cell proteases lysed casein and Azocoll but not native collagen. In low salt concentrations the enzymes existed at high molecular weight complexes, which were dissociated by increasing the salt concentration above 1.0 M (NaCl, KCl). Gel filtration in 1.4 M KCl separated the protease activity into three peaks, all of which activated procollagenase. Two of the enzymes showed substrate specificities (hydrolysis of p-tosyl-L-arginine methyl ester and benzoyl-tyrosine ethyl ester) and reactive center reactivities similar to pancreatic trypsin and chymotrypsin. Based on gel filtration, apparent molecular weights of 160 000 (p-tosyl-L-arginine methyl ester esterase), 90 000 (main procollagenase activator) and 36 000 benzoyl-tyrosine ethyl ester esterase) were determined. Activation of procollagenase resulted in a 18-20 000 decrease of the molecular weight. The activation was directly related to the amount of activator added within certain limits. Further addition of activator resulted in proteolytic inactivation of collagenase.
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PMID:Activation of fibroblast procollagenase by mast cell proteases. 5 9

The three-dimensional structures of the bacterial serine proteases SGPA, SGPB, and alpha-lytic protease have been compared with those of the pancreatic enzymes alpha-chymotrypsin and elastase. This comparison shows that approximately 60% (55-64%) of the alpha-carbon atom positions of the bacterial serine proteases are topologically equivalent to the alpha-carbon atom positions of the pancreatic enzymes. The corresponding value for a comparison of the bacterial enzymes among themselves is approximately 84%. The results of these topological comparisons have been used to deduce an experimentally sound sequence alignment for these several enzymes. This alignment shows that there is extensive tertiary structural homology among the bacteria and pancreatic enzymes without significant primary sequence identity (less than 21%). The acquisition of a zymogen function by the pancreatic enzymes is accompanied by two major changes to the bacterial enzymes' architecture: an insertion of 9 residues to increase the length of the N-terminal loop, and one of 12 residues to a loop near the activation salt bridge. In addition, in these two enzyme families, the methionine loop (residues 164-182) adopts very different comformations which are associated with their altered substrate specificities.
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PMID:Amino acid sequence alignment of bacterial and mammalian pancreatic serine proteases based on topological equivalences. 9 20

The direct oxidation of a dipeptide azlactone (1) by DDG affords an optically active unsaturated dipeptide azlactone (2). It was shown that the double bond had the Z-configuration and that no racemization of the proline moiety occurred during the oxidation. The dehydrotripeptide, Z-Pro-delta-Phe-Phe OH (9b) was prepared by direct coupling of azlactone (2) with phenylalanine tetramethyl-guanidinium salt and was shown to be stable to chymotrypsin. The tetrapeptide, Z-Pro-delta-Phe-His-Leu OH (11), was prepared directly from the azlactone (2) and by saponification of the tetrapeptide ester (10) prepared by mixed anhydride coupling of Z-Pro-deltaPhe OH (7) with the dipeptide ester. The dehydropeptide (11) inhibits angiotensin I converting enzyme (IC50, 1X10-4 M).
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PMID:The synthesis of N-benzyloxycarbonl-L-prolyl-dehydrophenylalanyl-L-histidyl-L-leucine: a converting-enzyme inhibitor. 21 95

Intact spermatozoa from rat cauda epididymis possess a Mg2+-dependent ATPase activity that hydrolyses externally added [gamma-32P]ATP. The ATPase reaction was linear with time for approx. 6 min and there was no detectable uptake of ATP by these cells. The ATPase activity of the whole spermatozoa was not due to leakage of the intracellular enzymic activity, contamination of the broken cells or any possible cell damage during incubation and isolation of spermatozoa. The activity of the enzyme was strongly inhibited (approx. 85%) by p-chloromercuribenzenesulphonic acid (50 microM) or the diazonium salt of sulphanilic acid (50 microM), which are believed not to enter the cells, whereas ouabain (0.5 mM), NaF (10 mM), NaN3 (2.5 mM) and oligomycin (5 microM) had no appreciable effect on the activity of the spermatozoal APTase. There was little loss of ATPase activity from the cells when washed with 0.5 mM-EDTA and an iso-osmotic or hyperosmotic medium. These data are consistent with the view that the observed ATPase activity is located on the external surface of spermatozoa. The sperm ecto-ATPase activity is resistant to the action of proteinases (50 micrograms/ml), namely trypsin, chymotrypsin and Pronase. Studies with various unlabelled phosphate esters indicate that the sperm ecto-ATPase is not a non-specific phosphatase and it has high degree of substrate specificity for ATP.
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PMID:Evidence for the occurrence of an ecto-(adenosine triphosphatase) in rat epididymal spermatozoa. 23 71

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

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.
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PMID:Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases. 24 Jul 15


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