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)

A high salt nuclear extract from the true slime mold Physarum polycephalum was used as a source of kinase activity for the incubation of calf thymus histones with [gamma-32P]ATP. A major proportion of the 32P incorporated into histones was acid-labile and alkali-stable. The nature of the alkali-stable phosphorylated component was analyzed by subjecting the phosphorylated protein to total alkaline hydrolysis and separating the resultant phosphoamino acids by anion exchange chromatography. The 32P-labeled material co-chromatographed with phosphohistidine standards and did not co-chromatograph with phosphoserine, phosphothreonine, or phosphotyrosine standards. In similar experiments using reversed phase high-performance liquid chromatography to separate the phosphoamino acids, the 32P-labeled phosphoamino acid behaved like the 1-isomer of phosphohistidine, in not being retained by the column, and unlike 3-phosphohistidine, phosphoserine, phosphothreonine, phosphotyrosine, and phosphoarginine, which were all retained on the column. Histone H4 was a good substrate for the histidine kinase activity and the location of the phosphorylated histidine residue was probed by peptide mapping using chymotrypsin or V8 protease. Both maps were consistent with labeling of histidine 75 and inconsistent with labeling of histidine 18. The data show that Physarum nuclei contain a major kinase activity which produces phosphohistidine. The methods we have developed for studying this kinase activity provide the basis for a complete characterization of the structure and function of the Physarum enzyme and can be applied to the study of similar kinase activities in other systems.
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PMID:Phosphorylation of histidine in proteins by a nuclear extract of Physarum polycephalum plasmodia. 406 4

The complementary strands of reovirus double-stranded ribonucleic acid (ds RNA) are synthesized sequentially in vivo and in vitro. In both cases, preformed plus strands serve as templates for the synthesis of the complementary minus strands. The in vitro synthesis of dsRNA is catalyzed by a large particulate fraction from reovirus-infected cells. Treatment of this fraction with chymotrypsin or with detergents which solubilize cellular membranes does not alter its capacity to synthesize dsRNA. The enzyme or enzymes responsible for dsRNA synthesis remain sedimentable at 10,000 x g after these enzyme or detergent treatments, indicating their particulate nature. Pretreatment of this fraction with ribonuclease, however, abolishes its ability to catalyze dsRNA synthesis, emphasizing the single-stranded nature of the template and its location in a structure permeable to ribonuclease. In contrast, the newly formed dsRNA is resistant to ribonuclease digestion at low salt concentrations and hence is thought to reside within a ribonuclease-impermeable structure.
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PMID:Mechanism of reovirus double-stranded ribonucleic acid synthesis in vivo and in vitro. 516 74

Kushner, D. J. (National Research Council, Ottawa, Ontario, Canada), and H. Onishi. Contribution of protein and lipid components to the salt response of envelopes of an extremely halophilic bacterium. J. Bacteriol. 91:653-660. 1966.-Removal of protein from envelopes of Halobacterium cutirubrum by peptic digestion left residues that required little or no salt for stability. The salt requirement of envelopes was also lowered by incubation in 0.1 m MgCl(2), and could be lowered even further by digestion with trypsin or chymotrypsin in 0.1 m MgCl(2). Dissolution of envelopes in low salt concentrations made their protein more susceptible to attack by these and other proteolytic enzymes. Removal of lipids raised the requirement for divalent cations, particularly for Mg(++); it slightly increased the Na(+) requirement and did not affect the requirement for K(+). It was concluded that the requirement for high salt concentrations in extreme halophiles is due to mutual repulsion between negatively charged groups on proteins rather than to repulsion between negatively charged phosphate groups on the lipids. The latter act primarily as sites on which divalent cations, especially Mg(++) which is required in high concentrations by growing cells, are bound. In this manner, the phosphate groups support envelope structure.
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PMID:Contribution of protein and lipid components to the salt response of envelopes of an extremely halophilic bacterium. 532 62

Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.
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PMID:The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin. 542 43

The hypothesis of a correlation between a 10 degrees-20 degrees C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12 degrees C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5 degrees C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0 degree C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11 degrees C, which may be due to a gel-liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.
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PMID:Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing. 618 Oct 83

Previous studies established that brain microsomes catalyze the transfer of [35S]sulfate from 3'-phosphoadenosine 5'-phospho[35S]sulfate to an O-linked oligosaccharide chain of a membrane glycoprotein and sulfamino groups of a membrane-associated proteoheparan sulfate (R. R. Miller and C. J. Waechter (1979) Arch. Biochem. Biophys. 198, 31-41). A large fraction of the proteoheparan [35S]sulfate can be released by treating the enzymatically labeled membranes from calf brain with 1 M NaCl. The salt-extracted 35S-labeled proteoglycan has been partially purified by a combination of ion-exchange and gel filtration chromatography. Based on chromatographic analyses, the 35S-labeled proteoglycan labeled in vitro is proposed to be a family of proteoheparan [35S]sulfates having an average molecular weight estimated to be 55,000. Variation in the length of the 35S-labeled polysaccharide chains partially accounts for the differences in molecular size of the proteoheparan [35S]sulfates. Binding studies reveal that the intact proteoheparan [35S]sulfates, as well as the free 35S-labeled polysaccharides released by mild alkali treatment, rapidly reassociate with calf brain membrane preparations. The association with calf brain membranes is saturable and reversible. Consistent with the binding being a specific interaction, only iduronic acid-containing glycosaminoglycans inhibit the association of the 35S-labeled proteoglycan with calf brain membranes and facilitate the disassociation. Neither the binding of the 35S-labeled proteoglycan to membranes nor the displacement was affected by hyaluronic acid, chondroitin 4-sulfate, or chondroitin 6-sulfate. The binding of the enzymatically labeled proteoheparan sulfate is reduced by preincubating membranes with either trypsin or chymotrypsin, but not with neuraminidase or phospholipase D. These results suggest that at least one class of proteoheparan sulfates could be specifically bound to one or more brain membrane proteins. The results also suggest a role for iduronosyl residues, and perhaps the stereochemical relationship of the carboxyl group to the O-sulfate moiety at C-2, in the recognition process.
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PMID:Structural features and some binding properties of proteoheparan sulfate enzymatically labeled by calf brain microsomes. 623 46

A procedure for dissociation of the nasal salt glands of the domestic duck, Anas platyrhynchos, into suspensions of individual cells has been developed. This technique employs enzymatic digestion with collagenase, hyaluronidase, and chymotrypsin; divalent cation chelation with EDTA; and gentle mechanical dispersion. Average cellular yields of 39 and 26% based on DNA recovered were obtained from the glands of freshwater- and saline-adapted ducks, respectively. Epithelial secretory cells comprised 60-80% of the cell suspensions with the remainder of the populations consisting of endothelial cells, fibroblasts, and blood cells. The dissociated cells were viable as judged by trypan blue exclusion (80-100%, maintenance of ultrastructural integrity, and retention of responsiveness to secretagogues and metabolic inhibitors. Methacholine chloride (0.5 mM) stimulated oxygen consumption by suspensions of both freshwater- and saline-adapted cells, whereas ouabain (0.05 mM) abolished the methacholine-stimulated respiratory response. These cell suspensions provide a promising system for the in vitro study of secretory mechanisms in the avian salt gland.
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PMID:Dissociation of avian salt gland: separation procedures and characterization of dissociated cells. 624 10

A recently developed photometric version of polyelectrolyte titration was applied for the determination of the number of charged residues on globular proteins. Based on the observation that oppositely charged polyelectrolytes form, in general, stoichiometric polyelectrolyte complexes, the protein solutions were incubated in excess with an oppositely charged polyelectrolyte, and the residual amount was back-titrated using o-toluidine blue for end point detection. It was found that within the range pH 2 to pH 9 the interaction of the polyelectrolytes, potassium polyvinylsulfate, polydiallylammonium chloride, and N-methylglycolchitosan iodide, with various proteins of known amino acid composition (ribonuclease A, trypsin, chymotrypsin A, pepsin, cytochrome c) occurs stoichiometrically through 1:1 ion pair interaction, irrespective of the spatial distribution of the interacting ionic sites. The close correspondence between the experimental data for the net charge and the calculated balance of ionized residues for the proteins at a given pH indicates that in the native structure of these proteins oppositely charged ionic functions are largely neutralized by the formation of intramolecular salt linkages. It is concluded that polyelectrolyte titration offers an easy access to the determination of the surface charge of proteins and other biopolymers. The data further support the notion of the importance of electrostatic cooperative interactions in biological systems.
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PMID:Charge determination of proteins with polyelectrolyte titration. 629 8

This paper describes the isolation, purification and properties of a specific inhibitor of calcium-activated neutral proteinase (CaANP) in rabbit skeletal muscle. The inhibitor was a thermo-acid-stable protein degraded by trypsin and chymotrypsin and seemed to contain two polypeptide chains with molecular weights of 70 000 and 13 000 daltons. Maximal inhibitory activity was obtained at neutral pH. High salt concentrations were needed to suppressinhibition. Inhibitor concentration had no effect on the optimal Ca++ ion levels for CaANP. These experiments also show that enzyme inhibitor association was instantaneous and did not need any incubation.
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PMID:Characterization and purification of a Ca2+ ion-activated neutral proteinase inhibitor in rabbit skeletal muscle. 629 43

We describe herein two different effects of protease inhibitors and substrates on receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) obtained from the intestinal mucosa of vitamin D-deficient chicks: inhibition of binding of 1,25(OH)2D3 to its receptor and stabilization of the receptor. Both L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, block [3H]1,25(OH)2D3 binding to the receptor. Fifty per cent inhibition of binding occurs at 20 microM TPCK, and 100% inhibition at 100-200 microM; TLCK is about 25-fold less effective. At higher concentrations (10-100 mM), the chymotrypsin substrates N alpha-p-tosyl-L-arginine methyl ester and tryptophan methyl ester and the cathepsin B inhibitor leupeptin also inhibit [3H] 1,25(OH)2D3 binding to its receptor. Different inhibitors and substrates interact with the receptor differently: TPCK (20 microM) and N alpha-p-tosyl-L-arginine methyl ester (10 mM) are reversible, noncompetitive inhibitors, L-tryptophan methyl ester (20 mM) is a reversible competitive inhibitor, and phenylmethylsulfonyl fluoride (300 microM) shows no effect on [3H]1,25(OH)2D3 binding to its receptor. The most stable form of unoccupied 1,25(OH)2D3 receptors from chick intestinal mucosa was that obtained from a low salt chromatin preparation (t 1/2 = 6.0 h). The presence of KCl drastically decreased receptor stability (t 1/2 = 1.8 h); and the addition of 2.5 mM CaCl2 further reduced their stability. Phenylmethylsulfonyl fluoride and Trasylol inhibited the KCl-induced receptor instability, but did not prevent the additional instability in the presence of CaCl2. In summary, TPCK and TLCK exert direct effects on the 1,25(OH)2D3 receptor molecule, independent of their protease inhibitor function. These compounds may prove useful as covalent affinity labels for the receptor. On the other hand, phenylmethylsulfonyl fluoride and Trasylol stabilize 1,25(OH)2D3 receptors, probably via inhibition of KCl-activated nuclear protease(s). This receptor stabilization will be advantageous in receptor assays and/or purification procedures.
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PMID:Differential effects of protease inhibitors on 1,25-dihydroxyvitamin D3 receptors. 631 80


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