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 kinetics of reduction of the b-type cytochromes in the electron transport particles (ETP) from Mycobacterium phlei were studied with nicotinamide adenine dinucleotide, reduced form (NADH) or succinate as electron donors. There appeared to be three active cytochromes b in the ETP,bS563 and bS559, which were reducible by either substrate, and bN563, which was reducible by NADH but not by succinate. In the presence of adenosine 5'-triphosphate, a substantial increase in b563 reduction was observed with succinate at anaerobiosis. This was followed by a decrease in absorption. Adenosine 5'-triphosphate did not effect an increase in cytochrome b563 reduction at transition with NADH, but the occurrence of a secondary decrease in absorption was reflected in a decrease in total enzymatic reduction. The adenosine 5'-triphosphate effect was altered in trypsin-treated ETP, and abolished by uncoupling agents or by removal of the coupling factor-latent adenosine triphosphatase. In the presence of a supernatant fraction obtained during the preparation of the ETP, b563 reduction with succinate was greatly increased. A smaller increase was observed with NADH. Cytochrome b reduction was also studied in ETP inhibited by 2-n-nonylhydroxyquinoline-N-oxide, which appears to inhibit at bS563. On the basis of these data the interrelationships among the b-type cytochromes can be described in relation to the M. phlei electron transport chain.
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PMID:Multiple forms of cytochrome b in Mycobacterium phlei: kinetics of reduction. 16 77

Neurospora glutamate dehydrogenase (NADP-specific) is rapidly inactivated upon reaction with tetranitromethane. This inactivation is completely prevented by the presence of coenzyme (NADP) or nicotinamide mononucleotide (NMN) but not by substrate. NADH, or 2'-monophosphoadenosine-5'-diphosphoribose. Amino acid analysis indicates that the primary effect of modification is nitration of a single residue of tyrosine per polypeptide chain. We have identified the reactive tyrosine by isolation of a single, uniquely labeled peptide after hydrolysis with trypsin followed by cleavage with cyanogen bromide. The modified residue proved to be tyrosine-168 in the linear sequence. This residue is not present in the part of the sequence that had been previously implicated as involved in the binding of the adenylate portion of the coenzyme. Both NMN and 2-monophosphoadenosine-5'-diphosphoribose act as competitive inhibitors of NADP in the oxidation of glutamate with Ki values of 4.65 x 10(-4) M and 4.30 x 10(-4) M, respectively. Thus, the specific protection afforded by NADP and NMN, but not by 2'-monophosphoadenosine-5'-diphosphoribose, indicates that tyrosine-168 is involved in binding the nicotinamide portion of the coenzyme.
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PMID:Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. III. Inactivation by nitration of a tyrosine residue involved in coenzyme binding. 23 46

Eight 2-(3,4-methylenedioxyphenyl)-5-arylamino1,3,4-oxadiazoles were synthesized, characterized by their sharp melting points, elemental analyses, and IR spectra, and evaluated for anticonvulsant activity. The protection afforded by oxadiazoles (100 mg/kg ip) against pentylenetetrazol (90 mg/kg sc)-induced convulsions ranged from 50 to 80%. All oxadiazoles inhibited the respiratory activity of rat brain homogenates during oxidation of pyruvate, alpha-ketoglutarate, and succinate. The presence of added nicotinamide adenine dinucleotide (NAD) to the reaction mixture during oxidation of pyruvate decreased the degree of inhibition. All oxadiazoles possessed antiproteolytic activity that was reflected by their ability to decrease trypsin-induced hydrolysis of bovine serum albumin. Such an inhibition was concentration dependent and ranged from 10.2 to 47.5 and from 15.7 to 71.8% by 0.5 and 1 mM oxadiazoles, respectively. All oxadiazoles competitively inhibited in vitro succinate dehydrogenase activity of rat brain homogenates.
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PMID:Anticonvulsant and antiproteolytic properties of 2,5-disubstituted oxadiazoles and their inhibition of respiration in rat brain homogenates. 66 May 24

Eight 5-(3,4-methylenedioxyphenyl)-3-arylaminomethyl-1,3,4-oxadiazole-2-thiones were synthesized, characterized by their sharp melting points, elemental analyses, and IR spectra, and evaluated for anticonvulsant activity. All substituted oxadiazole-2-thiones possessed anticonvulsant activity, which was reflected by their ability to provide 10--70% protection against pentylenetetrazol-induced convulsions in mice at 100 mg/kg ip. These compounds inhibited in vitro nicotinamide adenine dinucleotide (NAD)-dependent oxidation of pyruvate, alpha-ketoglutarate, and NADH by rat brain homogenates as well as NAD-independent oxidation of succinate by rat brain homogenates. Antiproteolytic activity of these substituted oxadiazole-2-thiones was reflected by their ability to inhibit trypsin hydrolysis of bovine serum albumin. These results indicated that the inhibition of cellular respiration and antiproteolytic activity of these substituted oxadiazole-2-thiones is not the biochemical basis for their anticonvulsant activity.
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PMID:Anticonvulsant and antiproteolytic properties of 3,5-disubstituted oxadiazole-2-thiones and their inhibition of respiration in rat brain homogenates. 71 83

The surface of streptococci presents an array of different proteins, each designed to perform a specific function. In an attempt to understand the early events in group A streptococci infection, we have identified and purified a major surface protein from group A type 6 streptococci that has both an enzymatic activity and a binding capacity for a variety of proteins. Mass spectrometric analysis of the purified molecule revealed a monomer of 35.8 kD. Molecular sieve chromatography and sodium dodecyl sulfate (SDS)-gel electrophoresis suggest that the native conformation of the protein is likely to be a tetramer of 156 kD. NH2-terminal amino acid sequence analysis revealed 83% homology in the first 18 residues and about 56% in the first 39 residues with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of eukaryotic or bacterial origin. This streptococcal surface GAPDH (SDH) exhibits a dose-dependent dehydrogenase activity on glyceraldehyde-3-phosphate in the presence of beta-nicotinamide adenine dinucleotide both in its pure form and on the streptococcal surface. Its sensitivity to trypsin on whole organism and its inability to be removed with 2 M NaCl or 2% SDS support its surface location and tight attachment to the streptococcal cell. Affinity-purified antibodies to SDH detected the presence of this protein on the surface of all M serotypes of group A streptococcal tested. Purified SDH was found to bind to fibronectin, lysozyme, as well as the cytoskeletal proteins myosin and actin. The binding activity to myosin was found to be localized to the globular heavy meromyosin domain. SDH did not bind to streptococcal M protein, tropomyosin, or the coiled-coil domain of myosin. The multiple binding capacity of the SDH in conjunction with its GAPDH activity may play a role in the colonization, internalization, and the subsequent proliferation of group A streptococci.
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PMID:A major surface protein on group A streptococci is a glyceraldehyde-3-phosphate-dehydrogenase with multiple binding activity. 150 Aug 54

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.
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PMID:Binding of NAD+ by cholera toxin. 282 99

Extraction of rat kidney cytosol with 10% charcoal at 4 C inactivated specific T3 binding. The decreased T3 binding in extracted cytosol could be restored by addition of boiled kidney cytosol. Three different factors (a, b, and c) which could increase T3 binding were identified by Sephadex G-50 column chromatography of boiled cytosol. Two factors (b and c) were eluted as relatively small molecules. Factor a was present in small amounts. Factor c was neutralized by incubation with EDTA, but factor b was not. Factor b was not destroyed by trypsin, protease, DNase, or RNase, but was destroyed by alkaline phosphatase. Factor b was destroyed by incubation with nicotinamide adenine dinucleotide phosphate (NADPH)-dependent glutathione reductase in the presence of oxidized glutathione. Although T3 binding to charcoal-extracted cytosol protein was not influenced by reduced glutathione or dithiothreitol, it was markedly increased by NADPH. Maximal activation induced by 50 microM NADPH was not further increased by further addition of endogenous factor b. The elution position of NADPH in gel chromatography corresponded to the elution position of factor b. Factor b or NADPH increased maximal binding capacity without changes in affinity constant. These observations suggest that T3-binding protein in cytosol is present in inactive and active forms and that the active form is generated by NADPH, which is present as one of the activators in cytosol. The effect of these cytosolic T3-binding proteins on nuclear T3 binding in vitro was also studied. In the absence of cytosolic T3-binding protein, [125I]T3 binding to nuclear receptor was decreased by unlabeled T3 in a concentration-dependent manner. In the presence of inactive form of cytosolic T3-binding protein, nuclear [125I]T3 binding was slightly diminished. In the presence of NADPH and cytosolic T3-binding protein, however, the amount of [125I]T3 bound to nuclei markedly decreased, which was associated with an increase of cytosolic [125I]T3 binding. NADPH alone did not influence nuclear T3 binding. These results suggest that T3 binding to nuclear receptor is regulated by an active form of cytosolic T3-binding protein in vitro.
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PMID:Active and inactive forms of 3,5,3'-triiodo-L-thyronine (T3)-binding protein in rat kidney cytosol: possible role of nicotinamide adenine dinucleotide phosphate in activation of T3 binding. 301 55

C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two separate protein components, designated components I and II, which individually have little activity, but, when mixed and treated with trypsin, exert the potent activity. The present study provides the evidence that component I of the toxin catalyzes the hydrolysis of NAD into nicotinamide and ADP-ribose, whereas component II does not, indicating that component I of C2T has NAD-glycohydrolase activity, which ability is shared with cholera and diphtheria toxins. However, C2T affected neither glycerol production of fat cells nor protein synthesis in cell-free system. Component I of C2T in the presence of [alpha-32P]NAD radiolabeled a protein of Mr 46,000 in the supernatant fractions of mouse tissue homogenates; the protein was abundant in brain, lung and intestine, whereas there was little or none of the protein in muscle. These results indicate that component I can catalyze the covalent attachment of the ADP-ribose moiety of NAD to intracellular protein, which differs from those modified with cholera and diphtheria toxins. The present data, together with previous findings, suggest that the biological activity of C2T is elicited by ADP-ribosylation activity of component I, which is internalized into the cells after binding to the receptor site introduced with the binding of component II to the cell surface membrane.
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PMID:NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin. 302 8

Nicotinamide 1,N6-ethenoadenine dinucleotide (etheno-NAD, epsilon-NAD), a fluorescent analogue of NAD, was able to serve as a substrate for the bacterial toxin-catalyzed epsilon-ADP ribosylation of signal-transducing G-proteins. Pertussis toxin and transducin were used as a model system to characterize this reaction. Similar to ADP ribosylation using NAD as substrate, the epsilon-ADP ribosylation occurs at the carboxyl-terminal 5-kDa tryptic fragment of the T alpha subunit of transducin with the same labeling stoichiometry; however, the rate of labeling is slightly slower. epsilon-NAD competes with NAD as a substrate which suggests that the epsilon-ADP ribosylation occurs at Cys-347 of the T alpha subunit. The biochemical effects of epsilon-ADP ribosylation on transducin are similar to those of ADP ribosylation and include inhibition of the GTPase and [3H]Gpp(NH)p-binding activities. The epsilon-ADP-ribosylated transducin exhibits a fluorescent spectrum which resembles that of epsilon-ADP with an excitation maximum at 292 nm and an emission maximum of 413 nm. Removal of the amino-terminal peptide of epsilon-ADP-ribosylated T alpha with either Staphylococcus aureus V8 protease or trypsin results in a decrease in the emission intensity. This result suggests that the amino- and carboxyl-terminal peptides of the T alpha molecule may interact with each other as suggested previously (Hingorani, V. N., and Ho, Y.-K. (1987) FEBS Lett. 220, 15-22). epsilon-NAD should prove to be a useful fluorescent substrate for future studies of the ADP ribosylation reaction in biological systems.
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PMID:Fluorescent labeling of signal-transducing G-proteins. Pertussis toxin-catalyzed etheno-ADP ribosylation of transducin. 314 31

Protease inhibitors are synthesized in biological systems and play a critical role in controlling a number of diverse physiological functions. They participate in blood clotting and lysis of clots, in growth processes by modulation of proteolytic digestion of proteins and thus availability of amino acids, and in the induction of selective DNA amplification. When incorporated into the diet, protease inhibitors appear to suppress many types of cancer. In vitro, they suppress neoplastic transformation caused by chemical carcinogens, ionizing radiation, and oncogenes. These observations offer the hope that judiciously applied protease inhibitors in small concentrations may prevent a wide range of human cancers. This hope is further supported by epidemiological studies which show that populations consuming relatively large amounts of protease inhibitors have a lower occurrence of cancer. The tasks remaining are to determine the kind and the level of protease inhibitors that are most effective in preventing cancer without also having toxic side effects and to incorporate them into our diet. Perhaps the most encouraging investigations are those using small nontoxic protease inhibitors available in pure form (epsilon-aminocaproic acid, a trypsin plasminogen activator inhibitor, and nicotinamide, a chymotrypsin inhibitor and known vitamin). Both agents have been shown to be preventive agents of cancer in animals and in vitro models. Further studies with natural protease inhibitors may yield even more effective agents which when incorporated into our diet will prevent the development of many types of cancer.
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PMID:Anticarcinogenic action of protease inhibitors. 331 95


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