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)

The role of cytosolic components in the regulation of mouse pancreatic islet adenylate cyclase activity was studied. Addition of mouse islet cytosol (27000 g supernatant of mouse islet sonicate), devoid of adenylate cyclase activity itself, increased adenylate cyclase activity by 93 +/- 17% (n = 9) in the 27000 g total particulate fraction of mouse islets. Addition of GTP stimulated adenylate cyclase activity by 91 +/- 11% (n = 13) or to the same degree as cytosol. Like GTP, the substance causing the enhancing activity of the cytosol was found to be dialysable, resistant to heat, sensitive to charcoal treatment and alkaline phosphatase and insensitive to digestion with trypsin. However, in contrast to the stimulation by GTP, the stimulation by cytosol was not inhibited by guanosine 5'-0-(2-thiodiphosphate), and furthermore, the effects of cytosol and GTP were additive. Neither NAD nor phosphoenolpyruvate stimulated adenylate cyclase activity. The cytosolic factor did not confer sensitivity towards glucose, Ca2+ or Ca2+-calmodulin on adenylate cyclase. The results demonstrate that mouse pancreatic islets contain a phosphocompound (or several compounds) distinct from GTP and capable of markedly stimulating adenylate cyclase. The identity of the compound and its physiological significance remain to be established.
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PMID:Characteristics of an adenylate cyclase enhancing factor from mouse pancreatic islet cytosol. 637 46

2-(5'-Dimethylaminonaphthalene-1'-sulfonamido)methylimidic acid methyl ester has been synthesized for fluorescence labelling of amino groups in proteins. The incorporation of the dansyl group serving as an extrinsic fluorescent probe is determined spectrophotometrically. Glucose dehydrogenase (beta-D-glucose: NAD(P+) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium having a reactive lysine residue which belongs to the active site has been labelled. To give proof of the selectivity of the modification, the enzyme preparation having 1.3 dansyl groups per subunit has been digested with trypsin and the major labelled peptide has been isolated and sequenced.
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PMID:Synthesis and application of a fluorescent imido ester for specific labelling of amino groups in proteins. 641 75

This work was done to discover how those nonphotosynthetic tissues of the Araceae that become thermogenic release, as CO2, carbon recently fixed by phosphoenolpyruvate carboxylase. Extracts of clubs of the spadix of Arum maculatum showed no activity for phosphoenolpyruvate carboxykinase and low activities of NADP malic enzyme. NAD malic enzyme activity in the above extracts and in those of thermogenic tissues of other Araceae was appreciable. Analysis of homogenates of clubs of Typhonium giraldii by differential centrifugation and sucrose gradients showed that NAD malic enzyme was confined to mitochondria. Centrifugation of mitochondria after freezing and thawing left all the NAD malic enzyme in the supernatant. NAD malic enzyme in isolated, intact mitochondria was completely latent, and was completely protected from exogenous trypsin. The responses of this latency and protection to different concentrations of Triton X-100 suggested that none of the NAD malic enzyme was accessible from either the outside or the intermembrane space of the mitochondria. Treatment of excised clubs of A. maculatum with 2-N-butylmalonate largely prevented the development of the rapid respiration responsible for thermogenesis, and severely inhibited dark fixation of 14CO2. The conclusion is that in mature clubs of the Araceae phosphoenolpyruvate is converted to malate in the cytosol by phosphoenolpyruvate carboxylase and NAD malate dehydrogenase, and that this malate then enters the mitochondrial matrix where it is converted to pyruvate by NAD malic enzyme.
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PMID:Role and location of NAD malic enzyme in thermogenic tissues of Araceae. 642 Dec 32

The amino acid sequence of the NADP+-dependent enzyme ovine 6-phosphogluconate dehydrogenase has been determined by conventional direct protein sequence analysis of peptides resulting from digestion of the protein with trypsin and chemical cleavages with cyanogen bromide, hydroxylamine, and iodosobenzoic acid. The polypeptide contains 466 amino acids and its NH2 terminus is acetylated. The Candida utilis enzyme is inactivated by reaction of pyridoxal phosphate with two lysine residues (Minchiotti, L., Ronchi, S., and Rippa, M. (1981) Biochim. Biophys. Acta 657, 232-242). These residues are conserved in the ovine enzyme. In contrast to NAD+ dehydrogenases which have weakly related sequences and spatially related folds in their nucleotide-binding sites, no significant sequence homologies were detected between 6-phosphogluconate dehydrogenase and any of three other NADP+-requiring enzymes, glutamate dehydrogenase, p-hydroxybenzoate hydroxylase, and dihydrofolate reductase. This is in accord with structural data that show no spatial relationship between NADP+-binding sites in these enzymes.
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PMID:Amino acid sequence of ovine 6-phosphogluconate dehydrogenase. 668 25

Cobaltous chloride induced in rat liver an enzyme which converted biliverdin reductase molecular form 1 into the molecular form 3. This conversion involves the oxidation of two sulfhydryl groups of form 1 giving rise to a disulfide bond in form 3. The converting enzyme was isolated from the liver peroxisomal fraction (which was devoid of biliverdin reductase activity), and was absent in liver peroxisomes of control rats. The enzyme was solubilized by treatment of the peroxisomes with 0.1% sodium deoxycholate, and partially purified by DEAE-cellulose and Sephadex G-100 filtration. It is a NAD+ dependent enzyme which was inactivated by trypsin and heat treatments. It did not oxidize either reduced glutathione or cysteine. The converting enzyme had a molecular weight of about 54,000 daltons. The oxidation of biliverdin reductase molecular form 1 mediated by the converting enzyme did not affect the latter's molecular weight or activity.
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PMID:Isolation from rat liver of a peroxisomal enzyme which converts molecular form 1 of biliverdin reductase into molecular form 3. 673 5

Supernatant fractions (300,000 x g, 60 min) from homogenates of rat liver, heart, and skeletal muscle, dog liver, and rabbit liver prepared without detergent in the homogenization medium (referred to as S300) are shown to contain an activity that restores Mg2+-dependent fluoride- and guanine nucleotide-stimulated cyclizing activity to the adenylate cyclase system [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] in cyc- S49 murine lymphoma cell membranes. Approximately 25% of the total cyc- reconstituting activity in the above tissues is present in S300. Reconstituting activity is proportional to S300, is sensitive to trypsin, is protected against heat inactivation by guanine nucleotide, and has a sedimentation coefficient of 5.3 in both H2O and 2H2O linear sucrose density gradients. Treatment with cholera toxin and NAD+ results in reconstitution of cyc- adenylate cyclase with enhanced activity in the presence of GTP. Reconstituion with S300 is stable, as seen in cyc- membranes after washing. All of these properties of S300 are similar to those of membrane-derived cyc- reconstituting activity. It is concluded that cell cytoplasm contains a naturally soluble protein or mixture of proteins having guanine nucleotide regulatory component activity of adenylate cyclase.
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PMID:Naturally soluble component(s) that confer(s) guanine nucleotide and fluoride sensitivity to adenylate cyclase. 693 40

1. Staphylococcal enterotoxin A (SEA) was exposed in a state of limited proteolysis to five kinds of proteolytic enzymes: papain, pepsin, pronase, trypsin and alpha-chymotrypsin. SEA was found to be sensitive to the action of three of them: papain, pepsin and pronase. 2. Four fragments were produced after papain proteolysis of SEA in the presence of beta-mercaptoethanol. 3. Papain processing of SEA does not influence its capacity to interact with antibodies to intact toxin, but the capacity of SEA to hydrolyse NAD to nicotinamide (NA) and adenosinediphosphatribose (ADP-ribose) completely disappears. 4. SEA under the action of pepsin and pronase has been hydrolysed to small peptides, which appear to be moving with the front of leading dye in disc-electrophoresis.
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PMID:Properties of staphylococcal enterotoxin A under limited proteolysis. 704 84

Incubation of crude normal rat kidney membranes with activated cholera toxin in the presence of DTP, ATP and NAD results in a 10--20 fold stimulation of adenylate cyclase activity. Optimal choleragen activation f the cyclase is shown to be dependent upon the presence of a plasma membrane-associated reconstituting activity, which can be dissociated from the membranes by washing with 10 mM potassium phosphate buffer, pH 7.5, containing 5 mM EDTA and 0.1 mM dithiothreitol. choleragen-catalyzed ADP ribosylation of plasma membrane substrate proteins also requires the presence of reconstituting activity factor. Sephadex G-150 filtration of solubilized reconstituting activity shows a peak activity eluting in the region corresponding to a protein with a molecular weight of approx. 13,000. Reconstituting activity is eluted from DEAE-cellulose at a salt concentration of 40--100 mM KCl. This active factor is not destroyed by trypsin treatment or by boiling for 30 min. These observations indicate that an endogenous membrane-associated factor, along with GTP, may be involved in modulating the ability of choleragen to activate adenylate cyclase.
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PMID:Plasma membrane-associated component(s) that confer(s) cholera toxin sensitivity to adenylate cyclase. 705 18

1. Sequence analysis of the NADPH domain (residues 158--293) and of the interface domain (365--478) was based on 12 CNBr fragments, which were isolated using ion-exchange chromatography and paper methods. Fragments with more than 15 residues were digested further with trypsin and chymotrypsin. The isolated peptides were sequenced by automated solid-phase Edman degradation. All sequenced peptides were ordered and overlapped by computerized comparisons with a complete sequence guessed from the electron density map of the protein. In the case of short CNBr fragments, this alignment was confirmed by the sequence analysis of protein fragments resulting from incomplete CNBr cleavage. 2. In the NADPH domain, residue 197, which is involved in an induced-fit mechanism, was identified as a tyrosine. The structure of the NADPH domain is probably homologous with the NAD domain of lipoamide dehydrogenase and with the FAD domain of several proteins, but not with NADPH domains of known chain-fold in other proteins. 3. The paper completes the sequence analysis of glutathione reductase so that the enzyme is now known in atomic detail. The numbering scheme of the chemically determined sequence will be used henceforth in crystallographic studies also. As inferred from the sequence data each of the two identical chains contains 478 amino acid residues, the composition being Cys10, Asp21, Asn17, Thr31, Ser31, Glu29, Gln11, Pro24, Gly43, Ala42, Val44, Met15, Ile29, Leu34, Tyr13, Phe14, Lys34, His16. Arg17, and Trp3. From these data an Mr of 2 x 51 600 was calculated for the FAD-free apoenzyme and an Mr of 2 x 42 400 for the holoenzyme.
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PMID:Glutathione reductase from human erythrocytes. The sequences of the NADPH domain and of the interface domain. 706 May 51

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43,000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13,000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD+, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.
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PMID:Activation of pigeon erythrocyte adenylate cyclase by cholera toxin. Partial purification of an essential macromolecular factor from horse erythrocyte cytosol. 721 14


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