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)

Treatment of submitochondrial particles (ETP) with trypsin at 0 degrees destroyed NADPH leads to NAD (or 3-acetylpyridine adenine dinucleotide, AcPyAD) transhydrogenase activity. NADH oxidase activity was unaffected; NADPH oxidase and NADH leads to AcPyAD transhydrogenase activities were diminished by less than 10%. When ETP was incubated with trypsin at 30 degrees, NADPH leads to NAD transhydrogenase activity was rapidly lost, NADPH oxidase activity was slowly destroyed, but NADH oxidase activity remained intact. The reduction pattern by NADPH, NADPH + NAD, and NADH of chromophores absorbing at 475 minus 510 nm (flavin and iron-sulfur centers) in complex I (NADH-ubiquinone reductase) or ETP treated with trypsin at 0 degrees also indicated specific destruction of transhydrogenase activity. The sensitivity of the NADPH leads to NAD transhydrogenase reaction to trypsin suggested the involvement of susceptible arginyl residues in the enzyme. Arginyl residues are considered to be positively charged binding sites for anionic substrates and ligands in many enzymes. Treatment of ETP with the specific arginine-binding reagent, butanedione, inhibited transhydrogenation from NADPH leads to NAD (or AcPyAD). It had no effect on NADH oxidation, and inhibited NADPH oxidation and NADH leads to AcPyAD transhydrogenation by only 10 to 15% even after 30 to 60 min incubation of ETP with butanedione. The inhibition of NADPH leads to NAD transhydrogenation was diminished considerably when butanedione was added to ETP in the presence of NAD or NADP. When both NAD and NADP were present, the butanedione effect was completely abolished, thus suggesting the possible presence of arginyl residues at the nucleotide binding site of the NADPH leads to NAD transhydrogenase enzyme. Under conditions that transhydrogenation from NADPH to NAD was completely inhibited by trypsin or butanedione, NADPH oxidation rate was larger than or equal to 220 nmol min-1 mg-1 ETP protein at pH 6.0 and 30 degrees. The above results establish that in the respiratory chain of beef-heart mitochondria NADH oxidation, NADPH oxidation, and NADPH leads to NAD transhydrogenation are independent reactions.
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PMID:Oxidation of NADPH by submitochondrial particles from beef heart in complete absence of transhydrogenase activity from NADPH to NAD. 0 Mar 95

Location of electron transport chain components in chloroplast membranes of chlamydomonas reinhardi, y-1 was investigated by use of proteolytic digestion with soluble or insolubilized trypsin. Digestion of intact membrane vesicles with soluble trypsin inactivates the water-splitting system, the 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition site of Photosystem II, the electron transport between the two photosystems as well as the ferredoxin NADP reductase. Reduction of NADP with artificial electron donors for Photosystem I could be restored, however, by addition of purified reductase to trypsin-digested membranes. Electron transfer activities of Photosystems I and II reaction centers were resistant to trypsin digestion either from outside or from within the thylakoids when active trypsin was trapped inside the membrane vesicles by sonication and digestion carried out in the presence of trypsin inhibitor added from outside. In the latter case, the water-splitting system was also found to be resistant to digestion. Polyacrylamide-bound insolubilized trypsin inactivated only the ferredoxin NADP reductase. Photosynthetically active membranes obtained at different stages of development showed a basically similar behavior toward trypsin.
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PMID:Trypsin-sensitive photosynthetic activities in chloroplast membranes from Chlamydomonas reinhardi, y-1. 0 Apr

The NADP-specific glutamate dehydrogenase of Neurospora crassa was digested with trypsin, and peptides accounting for 441 out of the 452 residues of the polypeptide chain were isolated and substantially sequenced. Additional experimental detail has been deposited as Supplementary Publication SUP 50052 (11 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem J. (1975) 145, 5.
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PMID:The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. The tryptic peptides. 0 Oct

Glutamate decarboxylase, gamma-aminobutyrate-alpha-ketoglutarate aminotransferase and NAD-linked and NADP-linked succinic semialdehyde dehydrogenase, all constituting the GABA (gamma-aminobutyrate)-shunt pathway of glutamate metabolism are localized in the mitochondrial matrix in a streptomycin-bleached mutant of Euglena gracilis strain Z. Glutamate dehydrogenase, requiring NADP as the cofactor, was distributed in the cytoplasm. An improved version of the controlled digestion method for preparing Euglena mitochondria, which involves use of trypsin and a trypsin inhibitor and removal of broken cells before mechanical disruption of cells, is also described.
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PMID:Subcellular localization of the GABA-shunt enzymes in Euglena gracilis strain Z. 11 50

HMG CoA reductase, which catalyzes the reaction, HMG CoA + 2 NADAPH2 leads to mevalonate + CoA-SH + 2 NADP, is considered to be the rate-limiting enzyme on cholesterol biosynthetic pathway. Since a degree in activity of this enzyme is almost proportional to the rate of cholesterol synthesis from acetate, elucidation of factors that regulate reductase activity would provide insight into the control mechanisms on the cholesterol biosynthesis. In the present study, attempts were made to establish standard assay conditions of HMG CoA reductase activiy, and to qualify the factors affecting the activity of the enzyme. The results obtained were as follows: (1) As standard assay conditions of HMG CoA reductase activity, 85, muM were chosen for substrate concentration, 25-80 mug for microsomal enzyme protein, and 20 min for incubation time in a final volume of 0.1 ml. (2) HMG CoA reductase activity of rat liver microsomes was exhibited diurnal variation. The level of reductase activity at night was 4 fold higher than that of at daytime. (3) Either ATP or insulin administration stimulated hepatic HMG CoA reductase activity. But, cyclic AMP had no effect on reductase activity. The stimulatory effect of ATP or insulin on reductase activity was inhibited by a preadministration of glucagon. These results suggested that an interplay of hormone might regulate reductase activity and consequently cholesterol biosynthesis. (4) HMG CoA reductase activity was increased by preincubation of microsomes with cytosol. Presence of ATP or Mg++ intensified this effect. When digested by trypsin or degenerated by heat treatment, cytosol lost the stimulating activity. These results suggested as existence of protein factors in cytosol, which might modulate the enzyme interconversion from inactive to active forms.
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PMID:[Studies on the regulatory factors of 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase) activity]. 18 33

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. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. Membrane stacking induced by Mg2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversible unstacked by brief treatment with trypsin, Mg2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, bt its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.
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PMID:Partition zone penetration by chymotrypsin, and the localization of the chloroplast flavoprotein and photosystem II. 44 96

NADPH-cytochrome P-450 reductase was isolated from liver microsomes of phenobarbital-induced rats. The enzyme exhibits an apparent minimal molecular weight of 76,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 molecule each of FMN and FAD. Trypsin treatment of the reductase yields an enzyme with an apparent minimal molecular weight of 69,000 which retains the ability to reduce cytochrome c but has no activity toward cytochrome P-450. Various spectrophotometric titrations were performed to examine the electron-accepting properties of the purified NADPH-cytochrome P-450 reductase and, in particular, to determine the oxidation state of the stable semiquinone form produced by air oxidation of NADPH-reduced enzyme. Titration of the air-stable semiquinone form of the reductase with ferricyanide indicated that 1 mol/2 mol of flavin was required for complete oxidation. Furthermore, a spectrum corresponding to that of the air-stable semiquinone form was produced by the addition of approximately 0.5 mol of reductant/2 mol of flavin when the oxidized enzyme was titrate with NADPH or dithionite under anaerobic conditions. The spectral changes which accompanied the overall reduction of oxidized enzyme to the reduced form with dithionite produced four sets of isosbestic points, and the spectrophotometric titration curve consisted of four approximately equal phases. In the titration with NADPH, no significant further reduction was observed after the addition of approximately 1.5 mol/2 mol of flavin. However, the enzyme was fully reduced by NADPH when an NAPH-generating system was used to prevent the accumulation of NADP. Our results establish that the air-stable semiquinone form is a 1-electron-reduced form, rather than a half-reduced (2-electron-reduced) form as maintained by others and are in agreement with earlier studies (Iyanagi, T., Makino, N., and Mason, H.S. (1974) Biochemistry 13, 1701-1710) with the purified trypsin-solubilized reductase. Accordingly, the air-stable species represents a form of the NADPH-cytochrome P-450 reductase in which one of the two flavins exists in the semiquinone state and the other in the oxidized state.
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PMID:Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states. 63 95

This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP-malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose-1,6-bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma-desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (M(r) = 11,564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%).
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PMID:Purification, properties and primary structure of thioredoxin from Aspergillus nidulans. 145 27

Binding of NADP to glucose-6-phosphate dehydrogenase (G6PD) from Dicentrarchus labrax liver has stabilized its native structure against thermal inactivation, guanidine hydrochloride unfolding and inactivation by tryptic digestion. The time-course of G6PD inactivation by guanidine hydrochloride in the presence of NADP has provided experimental evidence in favor of a conformational drift upon NADP binding to the bass enzyme. Based on the inactivation patterns obtained when the enzyme was treated with guanidine hydrochloride and trypsin, it is proposed that the enzyme conformation induced upon NADP binding is in slow equilibrium with the conformation stabilized in the absence of NADP. FPLC studies have shown that micromolar concentrations of NADP induced oligomerization of G6PD. In addition, the different K0.5 values of NADP binding to the enzyme, ranging from 1-2 microM (from trypsin inactivation) to 90 microM (from titration of the intrinsic fluorescence), suggest a step-wise binding of NADP to the oligomer, with negative cooperativity in the saturation process.
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PMID:Unfolding and trypsin inactivation studies reveal a conformation drift of glucose-6-phosphate dehydrogenase upon binding of NADP. 163 1


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