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)

Bovine liver and mammary UDP-galactose-4-epimerases were investigated with respect to various inhibitors and inactivators. Uridine nucleotides and NADH are potent inhibitors with Ki values in the low micromolar range. The NAD+/NADH ratio may be an important physiological control mechanism for it affects markedly the activity of the enzyme with 50% inhibition occurring at a ratio of 20:1. In the presence of uridine nucleotides binding of NADH to the epimerases is enhanced. Consequently, the effect of changes in the NAD+/NADH ratio in vivo would not be immediately apparent as uridine nucleotides would slow down the displacement of NADH by NAD+. Neither uridine nor galactose 1-phosphate inhibits the purified enzymes as previously reported with the impure liver enzyme. Uridine nucleotides provide almost total protection against the apparent first order inactivation of the epimerases by trypsin and allow determination of dissociation constants. NAD+ partially protects against trypsin inactivation. Inactivation with various sulfhydryl reagents is complex and the results indicate that at least three sulfhydryl groups may be modified before total inactivation occurs. Partial inactivation occurs upon modification of the epimerases with 2-hydroxy-5-nitrogenzyl bromide. Some protection against this modification is provided by the combination of NAD+ and UDP.
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PMID:Inhibition and inactivation of bovine mammary and liver UDP-galactose-4-epimerases. 19 53

Pyridine dinucleotide transhydrogenase of the Rhodospirillum rubrum chromatophore membrane was readily resolved by a washing procedure into two inactive components, a soluble transhydrogenase factor protein and an insoluble membrane-bound factor. Transhydrogenation was reconstituted on reassociation of these components. The capacity of the membrane factor to reconstitute enzymatic activity was lost after proteolysis of soluble transhydrogenase factor-depleted membranes with trypsin. NADP+ or NADPH, but neither NAD+ nor NADH, stimulated by several fold the rate of trypsin-dependent inactivation of the membrane factor. Substantial protection of the membrane factor from proteolytic inactivation was observed in the presence of Mg2+ ions, an inhibitor of transhydrogenation, or when the soluble transhydrogenase factor was bound to the membrane. Coincident with the loss of enzymatic reconstitutive capacity of the membrane factor was a loss in the ability of the membranes to bind the soluble transhydrogenase factor in a stable complex. The membrane component was inactivated by preincubating soluble transhydrogenase factor-depleted membranes at temperatures above 45 degrees. NADP+, NADPH, or Mg2+ ions, but neither NAD+ nor NADH, protected against inactivation. These studies indicate that (a) the binding of NADP+ or NADPH to the membrane factor promotes a conformational alteration in the protein such that its themostability and susceptibility to proteolysis are increased, and (b) the inhibitory Mg2+ ion-binding site resides in the membrane component.
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PMID:Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase. Proteolytic and thermal inactivation of the membrane component. 23 41

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.
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PMID:Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles. 23 83

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

1. It is shown by limited tryptic digestion of beef liver glutamate dehydrogenase under native conditions that the amino terminus of the polypeptide chain is located at the surface of the molecule. End-group analysis after trypsin treatment yields aspartic acid as the new N-terminal amino acid while the C-terminal threonine remains unchanged. 2. NADH, especially in the presence of 2-oxoglutarate, protects the enzyme against tryptic degradation. In the absence of the coenzyme, glutamate dehydrogenase is rapidly inactivated. 3. The regulatory effects of ADP and GTP are only slightly altered by trypsin. A small shift of the pH dependence of the activation by ADP is observed. 4. The quaternary structure of the unimer of the enzyme is not affected by limited tryptic digestion indicating that the N-terminal part of the polypeptide chain is not located in the contact domains between the polypeptide chains. The association of the hexamer to large associated particles is reduced but not abolished. 5. It is shown by treatment of the enzyme with iodo[2(-14)C]acetic acid as well as with Ellman's reagent that the six - SH groups of the polypeptide chain are buried and not accessible to these reagents in phosphate buffer. In Tris buffer they become exposed and react in the order 89, 55, 197, 115, 270, 319. This together with the result that in Tris buffer the rat of inactivation caused by trypsin is higher than in phosphate buffer indicates that Tris buffer changes drastically the properties of the enzyme. 6. Cross-linking of the enzyme molecule with bifunctional reagents and subsequent dodecylsulfate-polyacrylamide electrophoresis shows that the six identical polypeptide chains are arranged in two groups of three. 7. The implications of these results for the tertiary and quaternary structure of beef liver glutamate dehydrogenase are discussed.
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PMID:Studies of glutamate dehydrogenase: analysis of functional areas and functional groups. 24 Jun 78

Kinetic measurements indicate that the energy-independent transhydrogenation of 3-acetylpyridine-NAD+ by NADPH in membranes of Escherichia coli follows a rapid equilibrium random bireactant mechanism. Each substrate, although reacting preferentially with its own binding site, is able to interact with the binding site of the other substrate to cause inhibition of enzyme activity. 5'-AMP (and ADP) and 2'-AMP interact with the NAD+- and NADP+-binding sites, respectively. Phenylglyoxal and 2,3-butanedione in borate buffer inhibit transhydrogenase activity presumably by reacting with arginyl residues. Protection against inhibition by 2,3-butanedione is afforded by NADP+, NAD+, and high concentrations of NADPH and NADH. Low concentrations of NADPH and NADH increase the rate of inhibition by 2,3-butanedione. Similar effects are observed for the inactivation of the transhydrogenase by tryptic digestion in the presence of these coenzymes. It is concluded that there are at least two conformations of the active site of the transhydrogenase which differ in the extent to which arginyl residues are accessible to exogenous agents such as trypsin and 2,3-butanedione. One conformation is induced by low concentrations of NADH and NADPH. Under these conditions the coenzymes could be reacting at the active site or at an allosteric site. The stimulation of transhydrogenase activity by low concentrations of the NADH is consistent with the latter possibility.
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PMID:Steady-state kinetics and the inactivation by 2,3-butanedione of the energy-independent transhydrogenase of Escherichia coli cell membranes. 38 87

Microsomal squalene epoxidase has previously been solubilized with Triton X-100 and resolved into fractions, FA and FB, by DEAE-cellulose chromatography (Ono T. and Bloch K (1975) J biol. Chem. 250, 1571-1579). It has now been found that FB is identical with NADPH-cytochrome c reductase (denoted FPT, EC 1.6.2.3). Although both NADPH and NADH served as electron donors, the former was preferred for squalene epoxidase activity in the reconstituted system of FA and FB. FB is characterized by its ability to reduce cytochrome c by NADPH. In place of FB, partially purified FPT was tested for its ability to support squalene epoxidation in the presence of FA. A stepwise purification of the deoxycholate-solubilized FPT yielded an increase in specific FPT activity with a parallel increase in squalene epoxidase activity. Bromelain-solubilized FPT was less effective. Rabbit antisera preparations to the purified FPT solubilized with trypsin were shown to inhibit concomitantly FPT activity and squalene epoxidase activity. These observations support the concept that squalene epoxidation is primarily mediated via a flavoprotein, NADPH-cytochrome c reductase, and a terminal oxidase, squalene epoxidase, which is distinct from cytochrome P-450.
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PMID:Involvement of NADPH-cytochrome c reductase in the rat liver squalene epoxidase system. 40 52

1. The microsomal haem oxygenase activity induced by the administration of CoCl2 was found mainly in the smooth-surfaced microsomal fraction, whereas that of the untreated control animals was widely distributed in smooth-surfaced microsomal, rough-surfaced microsomal and Golgi fractions. 2. When microsomal preparation was incubated and the time course of the distribution of biliverdin between the membranes and the medium was followed, most of the biliverdin formed was found first in the medium. This suggests that the active site of haem oxygenase is exposed on the cytoplasmic surface of the membranes. The possible localization of the enzyme at the outer surface of the membranes was also supported by a digestion experiment with trypsin. The haem oxygenase activity was greatly decreased even at low concentration of the proteinase, which did not affected the NADPH-cytochrome c reductase activity. 3. When microsomal preparation was further fractionated by isopycnic centrifugation in the presence of deoxycholate or by partitioning of sonicated microsomal preparation in aqueous-polymer two-phase systems, most of the haem oxygenase activity was found in a fraction different from the main fraction of the NADH- and NADPH-cytochrome c reductase and NADH--ferricyanide reductase activities. This indicates the different distribution of haem oxygenase from the other enzymes mentioned, on the lateral plane of microsomal membranes, and suggests the different localization of the haem oxygenase system from the electron-transport system linked with cytochrome b5 and cytochrome P-450.
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PMID:Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride. 44 18

Intact microsomal vesicles from rat liver were subjected to combined treatment with trypsin and an unspecific protease and were also examined after reaction with the chemical probe p-diazobenzene sulfonate. In addition, the latency of various enzymes in intact microsomal vesicles has been investigated. All microsomal electron transport enzymes studied, i.e. NADH-ferricyanide and cytochrome c reductases, cytochrome b5, NADPH-cytochrome c reductase and cytochrome P-450, were either solubilized or inactivated by one or both treatments. The experimental data indicate that UDPglucuronyl-transferase is also localized at the outer surface of microsomes. In contrast, a number of hydrolytic enzymes are apparently located inside the permeability barrier of the membrane and presumably at the inner surface. Under conditions where the levels of electron transport enzyme activities or amounts are changed, such as in newborn rats and rats treated with phenobarbital or methylcholanthrene, the intramembranous position of these enzymes is the same as in control adult rats. This indicates that the enzyme molecules are not relocated after their insertion into the membrane.
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PMID:Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate. 66 58

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


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