Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.
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PMID:Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes. 17 49

Male rats were fed a cholesterol-free diet for 5 weeks, followed by a 2% cholesterol diet for 4 weeks. Another group of rats was continuously fed a cholesterol-free diet. A third group was fed standard pelllets during the whole experiment. Hepatic microsomal protein and cholesterol contents and drug-metabolizing enzyme activities were measured. The cholesterol-rich diet increased microsomal protein content and this increase disappeared after trypsin digestion of microsomal membranes. Microsomal cholesterol content was enhanced three-fold by cholesterol feeding. Cytochrome P-450 concentration, NADPH cytochrome c reductase and aryl hydrocarbon hydroxylase activities showed only minor changes following cholesterol feeding. The p-nitroanisole O-demethylase and ethoxycoumarin deethylase activities were doubled by cholesterol in comparison to cholesterol-free diet. Trypsin digestion activated the UDP-glucuronosyltransferase enzyme eight- to ten-fold on a protein basis. Trypsin treatment increased the cholesterol activation of UDP-glucuronosyltransferase when compared to the activity in native microsomes. The data suggest that dietary cholesterol regulates the cholesterol content of microsomal membranes. The activities of drug-metabolizing enzymes are also altered, possibly due to the compositional changes of the membranes.
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PMID:Dietary cholesterol-induced enhancement of hepatic biotransformation rate in male rats. 70 59

Developing pea (Pisum sativum L.) cotyledons were labeled with radioactive amino acids, glucosamine, and mannose in pulse an pulse-chase experiments to study the synthesis, glycosylation, and transport of the reserve proteins vicilin and legumin to the protein bodies. Tissue extracts were fractionated on sucrose gradients to isolate either the endoplasmic reticulum (ER) or the protein bodies. Immunoaffinity gels were used to determine radioactivity in the reserve proteins (legumin and vicilin). After pulse-labeling for 45 min with amino acids, about half the total incorporated radioactivity coincided closely with the position of the ER marker enzyme NADH-cytochrome c reductase at a density of 1.13 g . cm-3 on the sucrose gradient. Both radioactivity and enzyme activity shifted to a density of 1.18 g . cm-3 in the presence of 3 mM MgCl2 indicating that the radioactive proteins were associated with the rough ER. Approximately half of the incorporated radioactivity associated with the rough ER was in newly synthesized reserve protein and this accounted for 80% of the reserve protein synthesized in 45 min. Trypsin digestion experiments indicated that these proteins were sequestered within the ER. In pulse-chase experiments, the reserve proteins in the ER became radioactive without appreciable lag and radioactivity chased out of the ER with a half-life of 90 min. Radioactive reserve proteins became associated with a protein body-rich fraction 20-30 min after their synthesis and sequestration by the ER. Pulse-chase experiments with radioactive glucosamine and mannose in the presence and absence of tunicamycin indicated that glycosylation of vicilin occurs in the ER. However, glycosylation is not a prerequisite for transport of vicilin from ER to protein bodies. Examination of the reserve protein polypeptides by SDS PAGE followed by fluorography showed that isolated ER contained legumin precursors (Mr 60,000-65,000) but not the polypeptides present in mature legumin (Mr 40,000 and 19,000) as well as the higher molecular weight polypeptides of vicilin (Mr 75,000, 70,000, 50,000, and 49,000). The smaller polypeptides of vicilin present in vicilin extracted from protein bodies (Mr 12,000-34,000) were absent from the ER. The results show that newly synthesized reserve proteins are preferentially and transiently sequestered within the ER before they move to the protein bodies, and that the ER is the site of storage protein glycosylation.
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PMID:Role of the endoplasmic reticulum in the synthesis of reserve proteins and the kinetics of their transport to protein bodies in developing pea cotyledons. 706 59

Methyl sterol oxidase of microsomal synthesis of cholesterol from lanosterol is a mixed-function oxidase that is dependent upon reduced pyridine nucleotide. The methyl sterol oxidase, as well as NADH-cytochrome c reductase, in intact rat liver microsomes are inhibited by anti-cytochrome b5 immunoglobulin, but NADPH-cytochrome c reductase is not affected. There is a decreased time lag prior to onset of reoxidation of steady state levels of reduced cytochrome b5 when 4-methyl sterol oxidase substrates are present. Trypsin treatment of microsomes destroys cytochrome b5 with loss of methyl sterol oxidase activity. Activity is restored by addition of purified cytochrome b5 to trypsin-treated microsomes. Initial attempts to solubilize and purify 4-methyl sterol oxidase have been only partially successful due to the extreme lability of the oxidase. However, DEAE-cellulose column chromatography of a detergent extract of microsomes yields a fraction that contains the oxidase, lipids, and NADH-cytochrome b5 reductase but is free of cytochrome b5. Oxidation of 4 alpha [30-3H] methyl-5 alpha-cholest-7-en-3 beta-ol by methyl sterol oxidase in this isolated fraction can be fully restored by the addition of purified liver microsomal cytochrome b5. These results strongly support the suggestion that membrane-bound cytochrome b5 of rat liver microsomes is an obligatory electron carrier from NADH to 4-methyl sterol oxidase.
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PMID:Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol oxidase. 722 57

Previously, we have proposed that bovine adrenocortical mitochondrial adrenodoxin reductase may possess a domain structure, based upon the generation of two major peptide fragments from limited tryptic proteolysis. In the present study, kinetic characterization of the NADPH-dependent ferricyanide reductase activity of the partially proteolyzed enzyme demonstrates that Km(NADPH) increases (from 1.2 microM to 2.7 microM), whereas Vmax remains unaltered at 2100 min-1. The two proteolytic fragments have been purified to homogeneity by reverse-phase HPLC, and amino-acid sequence analysis unambiguously demonstrates that the 30.6 kDa fragment corresponds to the amino terminal portion of the intact protein, whereas the 22.8 kDa fragment is derived from the carboxyl terminus of the reductase. Trypsin cleavage occurs at either Arg-264 or Arg-265. Covalent crosslinking experiments using a water-soluble carbodiimide show that adrenodoxin crosslinks exclusively to the 30.6 kDa fragment, thus implicating the N-terminal region of adrenodoxin reductase in binding to the iron-sulfur protein. Our inability to detect covalent carbohydrate on either intact or proteolyzed adrenodoxin reductase prompted a re-examination of the previously reported requirement of an oligosaccharide moiety for efficient electron transfer from the reductase to adrenodoxin. Treatment of adrenodoxin reductase with a highly purified preparation of neuraminidase demonstrates that neither the adrenodoxin-independent ferricyanide reductase activity nor the adrenodoxin-dependent cytochrome c reductase activity of the enzyme is affected by neuraminidase treatment.
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PMID:Structural and functional characterization of bovine adrenodoxin reductase by limited proteolysis. 781 29

Bovine heart ubiquinol-cytochrome c reductase (bc1 complex) was modified with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ), which is a selective reagent for buried carboxyl groups. EEDQ treatment caused a loss of the proton pumping activity of liposome-reconstituted bc1 complex, without effect on the passive proton conductivity of the proteoliposomes. Although the decoupling effect produced on proton translocation was similar to that elicited by N,N'-dicyclohexylcarbodiimide (DCCD) modification of cytochrome b and subunit IX, EEDQ modified different subunits, namely the Core protein II and the iron-sulfur protein (ISP). A time-dependent increase of the labeling of both subunits was observed which was kinetically comparable with the decrease of the H+/e- ratio. Trypsin treatment of the complex showed that the EEDQ-modified carboxyl group in the ISP belongs to the protruding moiety of the protein, holding the Fe/S cluster. The results obtained show that critical acidic residues, located in different subunits of the bc1 complex, at both sides of the membrane, contribute to its proton pumping activity.
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PMID:Chemical modification of the bovine mitochondrial bc1 complex reveals critical acidic residues involved in the proton pumping activity. 948 30

The NAD(P)H-nitrate reductase complex (overall-NR) of Chlamydomonas reinhardii exhibits two partial activities: NAD(P)H-cytochrome c reductase (diaphorase) and reduced benzyl viologen-NR (terminal-NR). Mild tryptic digestion of the enzyme complex resulted in the loss of both overall and terminal-NR activities, whereas diaphorase activity remained unaltered. The diaphorase activity of mutant 104 and the terminal-NR activity of mutant 305 of C. reinhardii, which are the sole activities related to NR present in these mutants, responded to tryptic treatment to the same extent as the corresponding activities of the wild enzyme complex. Trypsin disassembled the 220-kd NR native complex by destroying the aggregation capability of the diaphorase subunits without affecting their activity nor molecular size (45 kd). A 67-kd thermostable protein, containing molybdenum co-factor, was also released from trypsin-treated NR. This protein lacked diaphorase and NR activities but was able to reconstitute the overall-NR complex by complementation with untreated diaphorase subunit of mutant 104. Our results support a tetrameric structure for the C. reinhardii NR complex, containing two kinds of subunits.
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PMID:Heteromultimeric structure of the nitrate reductase complex of Chlamydomonas reinhardii. 1645 30