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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of platelet release products on cytosolic calcium [( Ca++]i) was examined by monitoring the fluorescence of chick embryonic heart cells loaded with the fluorescent calcium indicator indo-1 AM. Cell free filtrate of platelet release products was obtained from rabbit platelets activated with thrombin or collagen. This filtrate caused a rapid increase in both systolic and diastolic [Ca++]i in a dose-dependent manner. The effect was not blocked by pretreating the platelets with aspirin or a thromboxane synthetase inhibitor. It was not mimicked by a thromboxane analog, or by several substances known to be released from platelets including ADP, serotonin, or platelet activating factor. Apyrase or ATP-gamma S had no effect on the activity. The responsible product was heat-sensitive, trypsin-sensitive, and partitioned into the aqueous phase of a chloroform suspension. It has a low molecular weight (less than 3kD) and is sensitive to 2-mercaptoethanol. Protease inhibitor appears to prolong the activity. These results suggest that trypsin-sensitive peptide(s) released from activated platelets can increase [Ca++]i in cardiac cells.
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PMID:Effect of platelet release products on cytosolic calcium in cardiac myocytes. 239 80

We have recently purified two proteins, alpha 39 and alpha 41, from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229). Both proteins bind guanine nucleotides and interact with beta.gamma units. We have used limited proteolysis by trypsin to probe the structure and the conformational states of these proteins. The guanosine 5'-O-(thiotriphosphate) (GTP gamma S)-liganded alpha 41 protein is cleaved into stable 39- and 24/25-kDa products which appear at the same rate. In addition, an 18-kDa peptide is seen. These products are also formed from GDP- or GTP-liganded alpha 41 but are less stable. Cleavage of alpha 39 is different. With GTP gamma S stable 37-kDa product predominates while with GTP or GDP the 37-kDa fragment appears transiently, followed by 24/25-kDa fragments which are stable in the presence of guanine nucleotides but rapidly cleaved without ligand. A 17-kDa peptide is also formed with GTP or GDP. The beta.gamma unit is cleaved by trypsin to stable peptides, a 26/27-kDa doublet and a 14-kDa peptide. Addition of beta.gamma slows tryptic cleavage of alpha 41 but not alpha 39. ADP-ribosylation of alpha 39 and alpha 41 by pertussis toxin affects their conformation in distinct ways which are clearly brought out by the GTP-liganded state. In contrast to unmodified alpha 41, ADP-ribosylated and GTP-liganded alpha 41 is proteolyzed very slowly and without formation of a 39-kDa intermediate. GTP gamma S seems to override the effect of ADP-ribosylation so that cleavage is more rapid and goes via the 39-kDa product. ADP-ribosylation affects alpha 39 more subtly. The GTP-liganded protein is first cleaved to the 37-kDa product and then degraded without forming the 24/25-kDa fragment. These results suggest that ADP-ribosylation might affect the conformation and function of these related proteins differently. The site of [32P]ADP-ribosylation is on the 18-kDa product of alpha 41 and on the 17-kDa product of alpha 39. We have raised polyclonal antibodies against alpha 39 and beta in rabbits and used the antibodies to examine antigenic sites on alpha 39 and beta. The antigenic determinants of alpha 39 are located over most of the native tryptic peptides. Tryptic cleavage of alpha 41 leads to rapid loss of cross-reactivity with anti-alpha 39 antibody.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Conformations of the alpha 39, alpha 41, and beta.gamma components of brain guanine nucleotide-binding proteins. Analysis by limited proteolysis. 242 23

Two mouse monoclonal IgM antibodies have been isolated which bind to histone 2B (H2B), as shown by protein blotting and immunostaining and by solid-phase radioimmunoassay (RIA). One of these (HBC-7) was specific for H2B by both techniques whereas the other (2F8) cross-reacted with histone H1 by RIA. Both antibodies failed to recognize H2B limit peptides from trypsin-digested chromatin and did not bind to Drosophila H2B, which differs extensively from vertebrate H2B only in the N-terminal region. These findings indicate that both antibodies recognize epitopes within the trypsin-sensitive, N-terminal region comprising residues 1-20. Binding of antibody HBC-7 was inhibited by in vitro ADP-ribosylation of H2B at glutamic acid residue 2. This strongly suggests that the epitope recognized by HBC-7 is located at the N-terminus of H2B, probably between residues 1 and 8. We have used solid-phase radioimmunoassay to investigate factors which influence the accessibility of this epitope in chromatin. Removal of H1 ('stripping') from high-molecular-mass chromatin had no effect on HBC-7 binding, nor was any difference observed between binding to stripped chromatin and to 146-base-pair (bp) core particles derived from it by nuclease digestion. These results suggest that accessibility of the N-terminal region of H2B is not influenced by H1 itself or by the size or conformation of linker DNA. In contrast, binding of antibody HBC-7 to 146-bp core particles derived from unstripped chromatin was reduced by up to 70%. Binding was restored by exposure of these core particles to the conditions used for stripping. Analysis of the protein content of core particle preparations from stripped and unstripped chromatin suggests that these findings may be attributable to redistribution of non-histone proteins during nuclease digestion. Pre-treatment of high-molecular-mass chromatin or 146-bp core particles with the intercalating dye ethidium bromide resulted in a severalfold increase in binding of HBC-7. The major changes in nucleosome morphology induced by ethidium are therefore accompanied by an increase in accessibility of the N-terminal region of H2B, possibly as a direct result of changes in the spatial relationship between H2B and core DNA.
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PMID:Characterization of monoclonal antibodies to histone 2B. Localization of epitopes and analysis of binding to chromatin. 242 56

Receptors for the 29-amino-acid peptide, galanin, in membranes from the rat ventral hippocampus were examined using chloramine-T-iodinated porcine galanin as ligand. The equilibrium binding of 125I-galanin showed the presence of a high-affinity binding site (Kd = 1.91 +/- 0.40 nM). The concentration of the high-affinity-binding sites was 107 +/- 15 fmol/mg membrane protein. The on rate constant was estimated to be 2.6 +/- 0.1 M-1 min-1 at 37 degrees C. The affinity of rat galanin (differing in three amino acid residues from the porcine protein) was equal to that of porcine galanin. The 125I--galanin-binding site is a trypsin-sensitive membrane protein, which is heat-denaturated at 60 degrees C within 5 min. The effect of GTP and its analogs and of pertussis-toxin-catalyzed ADP-ribosylation on the binding of 125I-galanin suggest that the galanin receptor is coupled to an inhibitory G protein (Gi protein). 127I-galanin was shown to be a ligand with affinity equal to that of galanin in displacing 125I-galanin. The 125I-galanin-binding site in the ventral hippocampus recognizes as a ligand the tryptic fragments 1-20 and 21-29 of rat galanin and the synthetic fragments 12-29, 18-29 and 21-29 of porcine galanin. None of these afforded full inhibition of the binding of fragment 1-29 of 125I-galanin at a concentration of 1 microM.
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PMID:Galanin receptor and its ligands in the rat hippocampus. 246 77

A new reactive fluorescent ADP analog has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 5'-diphosphate (2-BDB-T epsilon A-5'-DP). Rabbit muscle pyruvate kinase is inactivated by 200 microM 2-BDB-T epsilon A-5'-DP in a biphasic manner, with an initial loss of 75% activity followed by a slow total inactivation. The rate constants for both phases exhibit nonlinear dependence on reagent concentration, consistent with reversible formation of an enzyme-reagent complex (KI = 133 microM) prior to irreversible reaction. Loss of activity is prevented by substrates. The best protection against inactivation is provided by phosphoenolpyruvate (PEP), KCl, and MnSO4, suggesting that the reaction occurs in the region of the PEP binding site. Incorporation of 1.7 mol/mol enzyme subunit accompanies 90% inactivation by 200 microM 2-BDB-T epsilon A-5'-DP in 80 min. However, in the presence of PEP, KCl, and MnSO4, 1.0 mol of reagent is incorporated when the enzyme is only 14% inactivated. These results indicate that 2-BDB-T epsilon A-5'-DP reacts with two groups on the enzyme, one of which is at or near the PEP binding site. Incubation of pyruvate kinase with related nucleotide analogs lacking a 5'-diphosphate or a diketo group suggests that the diketo group, but not the diphosphate, is essential for inactivation. The enolized form of the bromodioxobutyl group resembles phosphoenolpyruvate and probably directs the reagent to the PEP binding site. Modified enzyme, prepared by incubating pyruvate kinase with 200 microM 2-BDB-T epsilon A-5'-DP in the absence and presence of phosphoenolpyruvate, KCl, and MnSO4, was reduced with [3H]NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on phenylboronateagarose followed by reverse phase high pressure liquid chromatography. Two radioactive peptides were identified: Asn162-Ile-Cys-Lys165 and Ile141-Thr-Leu-Asp-Asn-Ala-Tyr-Met-Glu-Lys150. Only the tetrapeptide was modified in the presence of PEP, KCl, and Mn+ when the enzyme retained most of its activity. Cys164 is thus designated the nonessential modified residue, while modification of Tyr147 near the active site of pyruvate kinase is responsible for loss of enzymatic activity. The observed biphasic kinetics of inactivation are due to the negatively cooperative reaction of 2-BDB-T epsilon A-5'-DP with Tyr147 in the tetramer. The new compound, 2-BDB-T epsilon A-5'-DP, may have general application as an affinity label of ADP and PEP sites in other proteins.
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PMID:2-[(4-Bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 5'-diphosphate. A new fluorescent affinity label of a tyrosyl residue in the active site of rabbit muscle pyruvate kinase. 248 27

Clostridium botulinum D (strain South Africa) produces ADP-ribosyltransferase which modifies eukaryotic 24-26-kDa proteins. ADP-ribosyltransferase activity was associated with a neurotoxin of 150 kDa (Dsa toxin) as confirmed by the elution profile of Dsa toxin from high performance anion-exchange column. The 24-kDa substrate of Dsa toxin-catalyzed ADP-ribosylation was detected in several tissues examined including rat brain, heart, and liver; bovine adrenal medulla; sea urchin eggs; electric organs of electric fish; and cell lines of neural (N18, N1E115, NS20Y, NG108, PC12, and C6) and non-neural (3T3) origins, suggesting its ubiquitous localization in eukaryotic cells. On the other hand, the 26-kDa substrate was detected only in membrane fractions of neural tissues and neuronal cells, suggesting its specific localization in membrane of nerve terminals. ADP-ribosylation of both the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane was potentiated by either divalent cations or guanine nucleotides, whereas adenine nucleotides did not affect the ADP-ribosylation reaction. Trypsin digestion of the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane extract produced different tryptic fragments indicative of the structural difference between the 24- and 26-kDa substrates. Both the 24- and 26-kDa substrates were less sensitive to trypsin digestion before being ADP-ribosylated by Dsa toxin than after, suggesting the conformational alterations of the 24-26-kDa proteins induced by ADP-ribosylation. These results suggest that Dsa toxin modifies two distinct low molecular mass GTP-binding proteins by ADP-ribosylation to alter their putative function(s).
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PMID:ADP-ribosylation of 24-26-kDa GTP-binding proteins localized in neuronal and non-neuronal cells by botulinum neurotoxin D. 249 19

We reported previously that the ADP-ribosyltransferase in C1 and D botulinum toxins specifically catalyzes ADP-ribosylation of an Mr 22,000 guanine nucleotide-binding protein and that this substrate named Gb (b = botulinum) has an amino acid sequence homologous to that deduced from the rho gene (Narumiya, S., Sekine, A., and Fujiwara, M. (1988) J. Biol. Chem. 263, 17255-17257). In this study we have determined the amino acid sequence at its ADP-ribosylation site. Purified substrate was [32P]ADP-ribosylated by C1 botulinum toxin and digested with trypsin. The radioactive peptides were isolated by reversed-phase high performance liquid chromatography and digested further either with protease V8, with proteases V8 and thermolysin, or with proline endopeptidase and thermolysin. By this procedure three radioactive peptides were obtained, and their amino acid sequences were X-Tyr-Val-Ala-Asp-Ile-Glu, X-Tyr, and Val-Phe-Glu-X-Tyr in which no amino acid peak was found in X. During the sequencing the radioactivity quantitatively adhered to the sequencing filter and was not eluted with either of the identified amino acid residues. Analysis of the protein without the ADP-ribosylation yielded the corresponding sequence as Thr-Val-Phe-Glu-Asn-Tyr which corresponds to Thr37-Tyr42 in the amino acid sequence deduced from the Aplysia rho gene. These results strongly suggest that the asparagine residue is the ADP-ribosylation site in the rho gene product. This ADP-ribose protein bond was stable in 0.5 M hydroxylamine at pH 7.5 at 37 degrees C for at least 5 h. The ADP-ribosylation of this protein affected neither its GTPase- nor its [35S]guanosine 5'-O-thiotriphosphate-binding activity.
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PMID:Asparagine residue in the rho gene product is the modification site for botulinum ADP-ribosyltransferase. 249 16

A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was [32P]ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture. The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP). No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP. Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP. Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP. When membrane Gi-alpha [32P]ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other. These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical. CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP. The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors. Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation. This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.
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PMID:Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells. 251 94

The role of blood platelets in the disturbed haemostasis in acute pancreatitis is not fully elucidated. The aim of this study was to evaluate the blood platelet function during the first hours of acute experimental pancreatitis (AEP) in dogs. AEP was induced by the retrograde injection of bile and trypsin into the main pancreatic duct. Platelet count, platelet aggregation induced with ADP, PAF, AA as well as plasma Beta-TG and TXB2 levels were determined. At 30 min after induction of AEP a significant decrease of platelet count was noted; these changes were observed until 4 th hr. At 30 min as well as at 60 min of AEP increased sensitivity of platelet aggregation to ADP was found. After that time evident decrease of platelet aggregation to ADP was shown. Platelets sensitivity to PAF was higher at 30 min of AEP whereas 60 min, 2 and 4 hrs after AEP normalization of platelet aggregation by PAF was observed. The significant increase of plasma Beta-TG and TXB2 concentrations corresponded well to changes of platelet aggregation. These results indicate that AEP affects blood platelet function with the drop of their count.
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PMID:Does acute experimental pancreatitis affect blood platelet function? 252 19

Conformational changes of 21 S dynein ATPase from sea urchin sperm flagella were examined by tryptic digestion under physiological conditions. In the presence of 2 mM ATP or ADP plus 100 microM inorganic vanadate (Vi), dynein heavy chains were digested by trypsin into quite different polypeptides from those obtained in other cases (no addition, 2 mM ATP, 4 mM adenosine 5'-(beta,gamma-imido)triphosphate, 4 mM adenosine 5'-(beta,gamma-methylene)triphosphate, 2 mM ADP, 100 microM Vi). In the presence of 4 mM adenosine 5'-O-(3-thiotriphosphate), however, the digestion pattern was similar to that in the presence of ATP (ADP) and Vi, to a certain extent. In all conditions other than the presence of ATP (ADP) and Vi, 165- and 135-kDa polypeptides were the main products, whereas in the presence of ATP (ADP) and Vi, 200-, 150/148-, and 105/96-kDa peptides were produced and 320-kDa peptide became rather inaccessible to trypsin. The latter digestion pattern was not observed in the absence of divalent cations. These results suggest that, in the ATP hydrolysis cycle, dynein changes its conformation remarkably in the dynein-ADP-Pi state, which is presumably responsible for force generation.
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PMID:Dynamic conformational changes of 21 S dynein ATPase coupled with ATP hydrolysis revealed by proteolytic digestion. 252 80


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