Reduced Nicotinamide Adenine Dinucleotide Phosphate-cytochrome Research Articles

A Professional and Personal Odyssey

As I began to write this article reflecting on my professional career in biomedical research, many memories of people, activities, situations, and experiences were swirling in my head. Because of the extraordinary support that I have received from my family, they are never far from my thoughts. I believe that sharing a few of these memories will be helpful for those who find themselves in similar situations or periods of professional development. This is not meant to be a comprehensive review of the fields to which I have had the privilege of contributing but a personal stroll down a path in which I have found extraordinary pleasure and from which I have derived a sense of accomplishment.

Cytochromes P-450scc, P-450(17)alpha, adrenodoxin, and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase in bovine follicles and corpora lutea. Changes in specific contents during the ovarian cycle.

To investigate the basis for the pattern of ovarian steroid production during the bovine estrous cycle, the tissue concentrations of major steroidogenic enzymes, 17 alpha-hydroxylase cytochrome P-450 and cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450scc), and their respective electron donors, NADPH-cytochrome P-450 reductase and adrenodoxin, were estimated and compared with those of the nonsteroidogenic enzymes, cytochrome c oxidase and F1-ATPase. The levels of these enzymes were estimated in medium sized (9-11 mm) and large (14-18 mm) follicles after removal of follicular fluid by centrifugation, and corpora lutea from the early, early-mid late-mid, and late stages of the luteal phase (n = 5 per group). The specific contents of all enzymes and electron donors were determined by immunoblot analysis, except for cytochrome c oxidase, which was quantified by determination of specific activity. The specific (per microgram of tissue homogenate protein) and total (per follicle or corpus luteum) tissue contents of 17 alpha-hydroxylase cytochrome P-450 increased 0.5- and 5-fold respectively from medium sized to large follicles, but then decreased to undetectable levels in corpora lutea in the early luteal phase, and remained undetectable throughout the luteal phase. In contrast, the specific content of NADPH-cytochrome P-450 reductase was similar between follicles and corpora lutea. The specific contents of cytochrome P-450scc, adrenodoxin and cytochrome c oxidase in follicles were similar to those of corpora lutea of the early luteal phase. However, by the early-mid luteal phase the specific contents of luteal cytochrome P-450scc (490 +/- 46 vs. 5709 +/- 982 cpm/micrograms protein) and adrenodoxin (44 +/- 15 vs. 705 +/- 229 cpm/micrograms protein) were increased, by 12- and 15-fold, respectively (P less than 0.05). In contrast, cytochrome c oxidase activity (29.1 +/- 10.1 vs. 108.6 +/- 20.7 nmol/mg tissue protein X min) and the specific content of F1-ATPase increased only 3- to 4-fold reflective of an increase in numbers of mitochondria. The levels of these enzymes remained elevated until the late luteal phase when they declined markedly. It is concluded that the induction of synthesis of P-450scc and adrenodoxin after ovulation is specific and does not merely reflect biogenesis of mitochondria during luteinization. Moreover the changes in the types of steroids produced by the ovarian compartments throughout the estrous cycle are a reflection of changes in the tissue content of steroidogenic enzymes.

Immunological and biochemical characterization of nuclear envelope reduced nicotinamide adenine dinucleotide phosphate-cytochrome c oxidoreductase

NADPH-cytochrome c oxidoreductase (EC 1.6.99.2) activity innate to rat liver nuclear envelope displays antigenic identity with the corresponding microsomal enzyme in a standard Ouchterlony double immunodiffusion test. As with the microsomal enzyme, the nuclear envelope enzyme is selectively released by restricted proteolysis and may be quantitatively isolated from the supernatant phase of the digest by immunoprecipitation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the immunoprecipitates reveals that the oxidoreductase has a molecular weight of 72,000 regardless of its membrane of origin. Radial immunodiffusion titration demonstrates that nuclear envelope contains about one-third the level of NADPH-cytochrome c oxidoreductase (0.21%) as compared to microsomal membrane (0.71%) on a weight basis. By comparison, the specific activity of the nuclear envelope enzyme was half that of the microsomal enzyme. Turnover studies employing NaH 14CO 3 indicate that the half-lives for the nuclear envelope and microsomal enzymes are indistinguishable, each being approximately 55 h.

Messenger ribonucleic acid metabolism in mammalian mitochondria. Isolation and characterization of polyribosomes from Ehrlich ascites mitochondria.

The Mg2+ precipitation procedure of R. D. Palmiter ((1974) Biochemistry 13, 3606) has been used for preparative scale isolation of polysomes from Ehrlich ascites mitochondria. Digitonin-washed metochondria used for isolating the polysomes contain no detectable reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and over 200-fold reduced hexokinase activity. The mitochondrial polysomes exhibit a heterogeneous sedimentation and appear to contain highly aggregated particlses ranging over hexamers. These polysomes are sensitive to RNase, (ethylenedinitrilo)tetraacetic acid and puromycin. Mitochondrial polysomes are active in portein synthesis when supplied with supernatant enzymes from the homologous mitochondrial source or from Escherichia coli. Cytoplasmic enzymes, however, appear to be completely inactive. Protein synthesis by mitochrondrial polysomes is sensitive to chloramphenicol and resistant to cycloheximide and emetine. The procedure yields particles containing intact rRNAs. The extent of cytoplasmic RNA contaminating the total mitochondrial RNA or mitochondrial polysomal RNA has been estimated to be negligible.

Evidence against multiple forms of reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase in rat liver microsomes

Evidence against multiple forms of reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase in rat liver microsomes

Nitroreduction of 5-nitrofuran derivatives by rat liver xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase

Rat liver cytosol and microsomes catalyzed the nitroreduction of N-[4-(5-nitro-2- furyl)-2-thiazolyl]acetamide (NFTA), a potent carcinogen for the mouse, rat, hamster and dog, and formed a metabolite capable of binding to protein. The cytosol nitroreductase was NADH or hypoxanthine dependent and strongly inhibited by a low concentration of allopurinol. Partial purification of the cytosol nitroreductase resulted in the parallel purification of nitroreductase and xanthine oxidase. Furthermore, NFTA, as well as 11 other nitrofurans, was reduced by purified milk xanthine oxidase. The metabolite formed was capable of binding to protein. These observations suggested that the cytosol nitroreductase activity was due to xanthine oxidase. The microsomal nitroreductase, which is NADPH dependent, was probably NADPH-cytochrome c reductase. Microsomal nitroreductase activity paralleled NADPH-cytochrome c reductase activity in rats pretreated with allylisopropylacetamide (AIA), phenobarbital or 3-methylcholanthrene (3-MC), but did not parallel the level of microsomal cytochrome P-450. A partial purification of the microsomal nitroreductase resulted in the parallel purification of nitroreductase and NADPH-cytochrome c reductase. The NFTA metabolite formed by the partially purified enzyme was capable of binding to protein. Other nitrofurans also were reduced by the same enzyme preparation. Hence, microsomal nitroreductase activity may be due to NADPH-cytochrome c reductase.

Preparation and Properties of Partially Purified Cytochrome P-450 and Reduced Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome P-450 Reductase from Rabbit Liver Microsomes

The liver microsomal enzyme system which hydroxylates fatty acids, hydrocarbons, and a variety of drugs and other foreign compounds was previously solubilized and resolved into three components: cytochrome P-450, NADPH-cytochrome P-450 reductase, and phospholipid. The two enzymes were partially purified from pyrophosphate-treated, cholate-solubilized liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000 and DEAE-cellulose column chromatography in the presence of Renex-690, a nonionic detergent. These steps were followed by treatment with Amberlite XAD-2 and calcium phosphate gel. Cytochrome P-450 preparations purified to a content as high as 15 nmoles per mg of protein were free of cytochrome b5 and contained no significant amount of NADPH-cytochrome c reductase, NADH-cytochrome c reductase, nonheme iron, other metals as measured by neutron activation analysis, or labile sulfide. The molecular weight was judged to be about 280,000 by gel exclusion chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis of the partially purified cytochrome P-450 preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of two major polypeptide bands, one with a molecular weight of 47,000 to 49,000 and the other 52,000 to 53,000. The component of lower molecular weight corresponded to the major polypeptide induced by phenobarbital treatment, as shown by electrophoresis of the proteins in normal and induced microsomes. The partially purified preparation accepted 2 electrons from dithionite per molecule of cytochrome P-450 under anaerobic conditions, indicating the presence of another electron acceptor in addition to the iron atom of the hemeprotein. The partially purified NADPH-cytochrome P-450 reductase preparations, which catalyzed the reduction of 8.6 to 10.0 µmoles of cytochrome c per min per mg of protein at 30°, were free of cytochrome P-450 and contained only a trace of NADH-cytochrome c reductase. FMN and FAD were found to be present in equimolar amounts, and labile sulfide was absent. The reconstituted enzyme system was active in the hydroxylation of fatty acids (laurate), hydrocarbons (hexane, cyclohexane, and octane), drugs (benzphetamine, hexobarbital, and ethylmorphine), and aniline. The presence of both phosphatidylcholine and deoxycholate was necessary for maximal activity.

Preparation of Partially Purified, Lipid-depleted Cytochrome P-450 and Reduced Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome c Reductase from Rat Liver Microsomes

Abstract Partially purified cytochromes P-450 and P-448 were obtained from hepatic microsomes of rats treated with either phenobarbital or 3-methylcholanthrene, using the ionic detergent sodium cholate and the nonionic detergent emulgen 911. The preparations contained 9 to 11 nmoles of cytochrome P-450 or P-448 per mg of protein and were free of NADPH-cytochrome c reductase and cytochrome b5. The total phospholipid content of the partially purified cytochrome P-450, per mg of protein, was reduced by more than 98% relative to microsomes. A partially purified NADPH-cytochrome c reductase preparation, essentially free of cytochrome b5 and phospholipid, is also described. Reconstitution of the cytochrome P-450 and reductase fractions with phospholipid resulted in restoration of enzymatic activity for the oxidative metabolism of a variety of substrates. Since the ratio of cytochrome P-450 to cytochrome b5 in this reconstituted system was greater than 200 to 1, it is suggested that cytochrome b5 is not an obligatory component of the mixed function oxidase system.

Formation of the red neotetrazolium formazan by reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase in the presence of triton X-100.

Formation of the red neotetrazolium formazan by reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase in the presence of triton X-100.

Microsomal electron transport: I. Reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and cytochrome P-450 as electron carriers in microsomal NADPH-peroxidase activity

An electron transport system that catalyzes the oxidation of NADPH by organic, hydroperoxides has been discovered in microsomal fractions. A tissue distribution study revealed that the microsomal fraction of rat liver was particularly effective in catalyzing the NADPH-peroxidase reaction whereas microsomes from adrenal cortex, lung, kidney, and testis were weakly active. The properties of the hepatic microsomal NADPH-peroxidase enzyme system were next examined in detail. The rate of NADPH oxidation by hydroperoxides was first-order with respect to microsomal protein concentration and a K m value for NADPH of less than 3 μ m was obtained. Examination of the hydroperoxide specificity revealed that cumene hydroperoxide and various steroid hydroperoxides were effective substrates for the enzyme system. Using cumene hydroperoxide as substrate, the reaction rate showed saturation kinetics with increasing concentrations of hydroperoxide and an apparent K m of about 0.4 m m was obtained. The NADPH-peroxidase reaction was inhibited by potassium cyanide, half-maximal inhibition occurring at a cyanide concentration of 2.2 m m. NADH was able to support the NADPH-dependent peroxidase activity synergistically. Evidence compiled for the involvement of NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) in the NADPH-peroxidase reaction included: (1) an identical pH optimum for both activities; (2) stimulation of NADPH-peroxidase activity by increasing ionic strength; (3) inhibition by 0.05 m m, p-hydroxymercuribenzoate with partial protection by NADPH; (4) inhibition by NADP +; and (5) inactivation by antiserum to NADPH-cytochrome c reductase. In contrast, antibody to cytochrome b 5 did not inhibit the NADPH-peroxidase activity. Evidence for the participation of cytochrome P-450 in the NADPH-peroxidase reaction included inhibition by compounds forming type I, type II, and modified type II difference spectra with cytochrome P-450; inhibition by reagents converting cytochrome P-450 to cytochrome P-420; and marked stimulation by in vivo phenobarbital administration. The NADPH-reduced form of cytochrome P-450 was oxidized very rapidly by cumene hydroperoxide under a CO atmosphere. It was concluded that the NADPH-peroxidase enzyme system of liver microsomes is composed of the same electron transport components which function in substrate hydroxylation reactions.

Kinetic studies on the control of ethylmorphine N-demethylase. The role of ethylmorphine activation of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTKinetic studies on the control of ethylmorphine N-demethylase. Role of ethylmorphine activation of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P 450 reductaseJordan L. Holtzman and Barry H. RumackCite this: Biochemistry 1973, 12, 12, 2309–2313Publication Date (Print):June 1, 1973Publication History Published online1 May 2002Published inissue 1 June 1973https://doi.org/10.1021/bi00736a019RIGHTS & PERMISSIONSArticle Views14Altmetric-Citations18LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (562 KB) Get e-Alerts Get e-Alerts

Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase.

Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase.

Studies on the microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase from rabbit liver.

Studies on the microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase from rabbit liver.

Isolation, Purification, and Some Properties of Reduced Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome c2 Reductase from Rhodopseudomonas spheroides

Abstract A method has been described for the isolation and purification of NADPH-cytochrome c2 reductase from light-grown Rhodopseudomonas spheroides. The enzyme is a nonmetalloflavoprotein with flavin adenine dinucleotide as the prosthetic group, and it catalyzes the reduction of R. spheroides cytochrome c2, 2,6-dichloroindophenol, and K3Fe(CN)6 with NADPH as the electron donor. It does not reduce R. spheroides cytochrome 553 but reduces Rhodospirillum rubrum cytochrome c2 and mammalian cytochrome c very slowly. There is no NADH or NADPH oxidase activity, NADP+ reductase activity, or transhydrogenase activity associated with the enzyme. The pH optimum was found to be 7.5; and Km values of 3.7 x 10-5 m, 1.25 x 10-5 m, and 1.24 x 10-4 m were calculated for cytochrome c2, 2,6-dichloroindophenol, and K3Fe(CN)6 reduction, respectively. NADH functioned as a competitive inhibitor, and the Ki was calculated to be 5.5 x 10-5 m. The enzyme was inhibited by p-chloromercuribenzoate, N-ethylmaleimide, iodoacetate, and thyroxine; and the inhibition by sulfhydryl reagents was reversed by reduced glutathione. Preincubation with NADPH protected the enzyme against the poisoning effects of sulfhydryl reagents but not thyroxine. The behavior of the enzyme on Sephadex G-100 and in the analytical ultracentrifuge indicates the presence of several molecular forms of the enzyme.

Studies on the mechanism of action of mammalian hepatic azoreductase—I: Azoreductase activity of reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase

Microsomal azoreductase was solubilized by steapsin, fractionated with ammonium sulfate, and purified by chromatography on DEAE-cellulose. The DEAE-cellulose eluate fraction was subjected to electrophoretic and ultracentrifugation analysis. The absorption spectrum of the purified enzyme was typical of a flavoprotein. From flavin determinations, a molecular weight between 33,000 and 45,000 was calculated. At no stage of purification was it possible to separate azoreductase and NADPH-cytochrome c reductase activities. Kinetic studies with purified enzyme demonstrated that the K m(NADPH) was of the same order of magnitude for the reduction of cytochrome c and of neoprontosil. The of the same order of magnitude for the reduction of cytochrome c and of neoprontosil. The enzyme could not be saturated with neoprontosil and K m (neoprontosil) was extremely high. Azoreductase and NADPH-cytochrome c reductase activities displayed similar patterns with respect to inhibition of sulfhydryl group inhibitors, and both activities showed similar heat lability. It was concluded that purified azoreductase is probably identical with purified NADPH-cytochrome c reductase. Microsomal azoreductase activity decreased upon solubilization, whereas NADPH-cytochrome c reductase activity increased, indicating the existence of a secondary azoreductase pathway or removal of a cofactor. In intact microsomes the reduction of the azo-linkage may not be entirely attributed to NADPH-cytochrome c reductase.

The Turnover of Microsomal Reduced Nicotinamide Adenine Dinucleotide Phosphate-Cytochrome c Reductase in the Livers of Mice Treated with Phenobarbital

Abstract When mice received injections twice daily of sodium phenobarbital there was a 2- to 4-fold increase in microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase within 4 days. In order to measure the turnover of the enzyme, tritiated l-leucine was injected 24 hours prior to treatment. The half-time for loss in vivo of radioactivity from enzyme protein averaged 2.8 days in controls, but there was no loss of radioactivity from the enzyme in phenobarbital-treated animals. There was little change in specific activity, since decreased breakdown in treated animals was balanced by increased synthesis of unlabeled protein. The 90-min incorporation of l-leucine-3H into microsomal NADPH-cytochrome c reductase was increased 2- to 3-fold in mice pretreated for 2 or more days with sodium phenobarbital. The accumulation of microsomal NADPH-cytochrome c reductase in animals treated with barbiturates seems to result from both a decreased rate of breakdown and an increased rate of synthesis.