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)

Acetylated derivatives of glucagon have been prepared by reacting this hormone under various conditions with acetic anhydride. They have been chemically characterized by the use of a 14C-labeled reagent, by peptide mapping techniques following hydrolysis by pronase and chymotrypsin, and by spectroscopy. Acetylation in sodium acetate (pH 5.5) results in a full substitution of the alpha-amino group of the N-terminal histidyl residue, but in a partial (about 0.3 acetyl group per residue) substitution of the epsilon-amino group of the lysyl residue 12. The monosubstituted (on the alpha-amino group) and the disubstituted (on both amino groups) acetylated components have been separated by chromatography on DEAE-cellulose and CM-cellulose. Acetylation in sodium bicarbonate (pH 8.0) results in a complete substitution of both amino groups and of the hydroxyl groups of the tyrosyl residues 10 and 13. Complete deacetylation of the O-acetyltyrosyl residues occurs upon treatment with hydroxyl-amine. Mono, di and tetraacetylglucagon are homogeneous when analyzed by disc gel electrophoresis; di and tetrasubstituted derivatives show an increased mobility towards the anode. 125I-labeled derivatives of acetylglucagon show higher distribution coefficients in the aqueous two-phase dextran/poly(ethylene glycol) system than do similar derivatives of glucagon. Acetylation decreases in parallel the ability of glucagon to stimulate the activity of adenylate cyclase and to bind to its receptors in liver cell membranes of the rat. The biological potencies of the mono, di and tetrasubstituted derivates are, respectively, about 10, 1 and 0.1% that of native glucagon. The binding properties of the material dissociated from the acetylglucagon-receptor complex suggest that the reduction in biological activity results from a decrease in the intrinsic affinity of the modified glucagon for the receptors, as well as from the presence of small amounts of residual, unreacted glucagon. Studies with 125I-labeled derivatives of glucagon indicate that acetylation decreases the rate of association and increases the rate of dissociation of the hormone-receptor complex.
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PMID:Acetylglucagon: preparation and characterization. 0 Dec 70

Human red blood cells (HRBC) even without prior neuraminidase treatment, could form rosettes with human peripheral blood lymphocytes in vitro. The optimum conditions for forming these rosettes were a pH of 7-0 and a medium with 5% bovine serum albumin (BSA). Rosette proportions became much less at a different pH or using lower concentrations of BSA, or replacing BSA with foetal calf sera (FCS) or human sera. Rosette formation was also promoted by prior treatment of HRBC or lymphocytes with neuraminidase. Mixed rosettes of HRBC and sheep red blood cells (SRBC) showed that HRBC receptors were detectable only on lymphocytes that possessed SRBC receptors, suggesting that HRBC rosette-forming cells were probably thymus-derived (T) cells. Next, the properties of human red blood cell (HRBC) and sheep red blood cell (SRBC) rosette-forming cells were investigated by comparing the ability of human peripheral blood lymphocytes to form these two types of rosettes after treatment with various inhibitory reagents. HRBC rosettes were relatively more resistant to inhibition with: (1) proteolytic agents, such as trypsin, alpha-chymotrypsin and pronase; (2) anti-thymocyte serum (ATS); (3) metabolic inhibitors, such as sodium azide and 2,4-dinitrophenol (DNP); (4) cytochalasin B. On further incubation after trypsinization, the lymphocytes recovered some ability to form SRBC rosettes, but continued to lose more of their capability to form HRBC rosettes. All these results were regarded as circumstantial evidence that the HRBC rosettes might represent a subpopulation of human T lymphocytes.
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PMID:Lymphocyte subpopulations. Human red blood cell rosettes. 0 4

1. alpha 1-Proteinase inhibitor was isolated from human plasma by a five-step procedure. Isoelectric focusing showed that six components focused between pH4.85 and 4.95. 2. The mol.wt. of the inhibitor was 52000 by sedimentation equilibrium and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid and carbohydrate compositions of the inhibitor were also determined. 3. The far-u.v.c.d. (circular-dichroism) spectrum indicated that the inhibitor had about 36% alpha-helical content. 4. The loss of proteinase-inhibitory activity when the inhibitor was exposed to pH values less than 5.0 or greater than 10.5 was accompanied by small changes in the far-u.v.c.d. spectrum and large changes in the near-u.v.c.d. spectrum. The change at alkaline pH was associated with ionization of tyrosine residues. 5. Interaction of inhibitor with chymotrypsin caused perturbation of the c.d. spectrum and this was used to follow the interaction and show a 1:1 stoicheiometry. 6. C.d., electrophoresis and isoelectric focusing showed that the inhibitor-enzyme complex is degraded by free enzyme. 7. Parallel studies with trypsin indicated that it too forms a 1:1 complex with inhibitor and is degraded by excess of enzyme.
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PMID:Isolation and characterization of human plasma alpha 1-proteinase inhibitor and a conformational study of its interaction with proteinases. 0 69

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

The self-association of alpha-chymotrypsin and its di-isopropyl phosphoryl derivative in in I0.03 sodium phophate buffer, pH7,9, was investigated by velocity sedimentation, equilibrium sedimentation and difference gel chromatography. No differences between the native and chemically modified enzyme were observed in the ultracentrifuge studies, and only a marginal (0.6%) difference in weight-average elution volume was detected by difference gel chromatography of 5g/litre solutions on Sephadex G-75. From quantitative analyses of sedimentation velocity and sedimentation-equilibrium distributions obtained with iPr2P (di-isopropylphosphoryl)-chymotrypsin, the polymerizing system is postulated to involve an indefinite association of dimer (with an isodesmic association constant of 0.68 litre/g) that is formed by a discrete dimerization step with equilibrium constant 0.25 litre/g. In addition to providing the best fit of the experimental results, this model of chymotrypsin polymerization at low ionic strength is also consistent with an earlier observation that dimer formation is a symmetrical head-to-head phenomenon under conditions of higher ionic strength (I0.29, pH7.9) where association is restricted to a monomer-dimer equilibrium. It is proposed that the dimerization process is essentially unchanged by variation in ionic strength at pH7.9, and that higher polymers are formed by an entirely different mechanism involving largely electrostatic interactions between dimeric species.
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PMID:Self-association of alpha-chymotrypsin at low ionic strength in the vicinity of its pH optimum. 1 54

A detailed examination of the mechanism of the hydrolysis of phenyl acetates by alpha-chymotrypsin [EC 3.4.21.1] was carried out. The effective deacylation rate constants of some phenyl acetates obtained by titration of the acetyl-enzyme decreased at low substrate concentrations and showed anomalous pH dependences and solvent isotope effects. The transient kinetics of deacylation of the acetyl-enzyme were biphasic. A spectrum and a breakdown rate similar to those of acetylimidazole were observed when the acetyl-enzyme was denaturated with sodium dodecyl sulfate. These results indicate the participation of histidine-acylated enzyme, which woud account for the anomalous phenomena previously found in this system, including a large value of Hammett's rho. The relation between the substrate activation and the two intermediates is discussed.
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PMID:alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetate. Large Hammett's rho constant and participation of histidine-acylated intermediate. 1 53

The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.
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PMID:Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the UDPglucose:glycogen 4-alpha-glucosyltransferase. 2 41

A proteolytic enzyme, which causes the limited degradation of cardiac myosin, was purified from rat heart myofibrils. The purified enzyme (a myosin-cleaving protease) was apparently homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Autolysis of the purified enzyme was observed at neutral pH without high concentration of CaCl2. The molecular weight was estimated to be 26 000-27 000. The enzyme was active against casein, N-acetyl-L-tyrosine ethyl ester and N-glutaryl-L-phenylalanine-4-nitroanilide (Glu-Phe-NAn), but less active with N-benzoyl-DL-arginine-4-nitroanilide. Optimum pH values for the enzyme were 9.0 for casein and 8.4 for Glu-Phe-NAn. Caseinolytic activity of the enzyme was completely inhibited with phenylmethylsulfonyl fluoride and diisopropylphosphofluoride and partially inhibited with L-1-tosyl-L-phenylalanine chloromethyl ketone (Tos-PheCH2Cl) and soybean trypsin inhibitor. Tos-LysCH2Cl had no effect. Sulfhydryl reagents, metal-chelating agents and metal ions except for Zn2+ had little or no effect on the activity. Degradation of cardiac myosin with the enzyme produced two fragments having molecular weights of 130 000 and 94 000, accompanied by the disappearance of myosin heavy chain and light chain 2. Myosin degradation with the enzyme was more restrictive than with chymotrypsin.
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PMID:Purification and characterization of a myosin-cleaving protease from rat heart myofibrils. 2 66

At pH 5.5, sodium trifluoroacetate is a potent competitive inhibitor of porcine elastase (Ki = 2.6 mM) and human leukocyte elastase (Ki = 9.3 mM). For both enzymes the Ki increases strongly with pH. Sodium fluoride is inactive on pancreatic elastase and sodium acetate is a weak inhibitor of this enzyme. Trifluoroethanol inhibits both enzymes but is less active than trifluoroacetate in acidic pH conditions. Bovine trypsin and alpha-chymotrypsin are resistant to the action of sodium trifluoroacetate and trifluoroethanol. The interaction between sodium trifluoroacetate and pancreatic elastase is also demonstrated by 19F NMR spectroscopy. Trifluoroacetyltrialanine is able to displace trifluoroacetate from its complex with pancreatic elastase. In addition, a method using turkey ovomucoid for the active site titration of leukocyte and pancreatic elastase is described.
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PMID:NMR and enzymatic investigation of the interaction between elastase and sodium trifluoroacetate. 2 76

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


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