Nicotinamide Adenine Dinucleotide Phosphate-cytochrome P-450 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.

Peri- and postnatal changes in reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase content in hepatocytes of rats.

To study the process of the expression of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase (EC 1.6.2.4) in the liver during development, the amount of enzyme in the cytoplasm of periportal and perivenular hepatocytes in sections cut from livers of male rats was measured during peri- and postnatal growth by quantitative immunohistochemistry with a video image processor. In livers of 19-day-old foetuses, the reductase content in the cytoplasm of periportal and perivenular hepatocytes was 0.16 microM and 0.20 microM, respectively. From the 19th day of gestation to 5 days after birth, the enzyme content increased markedly in the cytoplasm of periportal (288%) and perivenular hepatocytes (301%). Subsequently, the content in the cytoplasm of periportal hepatocytes increased slightly (46%) from 5 to 20 days of age, remained unchanged from 20 to 45 days of age, and increased slightly (15%) from 45 to 90 days of age. However, the content in the cytoplasm of perivenular hepatocytes increased progressively (125%) between 5 and 90 days of age. Thus, the amount of cytochrome P-450 reductase increases markedly in periportal and perivenular hepatocytes during the perinatal period, and subsequently the enzyme content increases gradually in periportal hepatocytes and progressively in perivenular hepatocytes. The present results also suggest that the divergence between cytochrome P-450 expression and the cytochrome P-450-dependent drug metabolic activity in hepatocytes during the perinatal period, found in previous studies, can be attributed to a low cytochrome P-450 reductase density in the membrane of endoplasmic reticulum of periportal and perivenular hepatocytes.

The role of iron chelators and oxygen in the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 oxidoreductase-dependent chromium(VI) reduction.

Chromium(VI) reduction was studied in a system composed of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P450 oxidoreductase (NADPH-P450 reductase) and different iron chelators and iron sources. In an aerobic phosphate buffer containing iron(II), chromium(VI) was not reduced by the Fe2+ probably because of spontaneous autoxidation of Fe2+, but freshly made Fe2+, added directly to a CrVI-containing buffer, reduced CrVI. Under anaerobic conditions, iron(II) reduced chromium(VI) stoichiometrically. A systemic containing ethylenediaminetetraacetic acid (EDTA)-Fe3+, NADPH-P450 reductase and NADPH effectively reduced chromium(VI) anaerobically. Under aerobic conditions this reaction was inhibited by about 45%. Adenosine diphosphate (ADP)-Fe3+, which is a poor acceptor of electrons from NADPH-P450 reductase, reduced chromium(VI) only marginally, Mannitol slightly increased the aerobic CrVI reduction. Addition of superoxide dismutase and catalase, which both regenerate some O2, led to inhibition of CrVI reduction. Ferritin, NADPH-P450 reductase and the iron chelators, EDTA and citrate, reduced CrVI, indicating mobilization of Fe2+ from ferritin. Low levels of EDTA (55 mumol l-1) and citrate (100 mumol l-1) in contrast to high levels (5 mmol l-1) did not increase CrVI reduction in microsomes. Using 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid buffer instead of phosphate buffer, the CrVI-reducing activity was increased.

Cross-linking studies of cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase.

Cross-linking studies of cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase.

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 455 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17α-hydroxyprogesterone in the presence of cytochrome P-450 21 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17α-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min

Destruction of heme and hemoproteins mediated by liver microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase.

Destruction of heme and hemoproteins mediated by liver microsomal reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 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.

Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b5 reductases.

Redox properties of the reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 and reduced nicotinamide adenine dinucleotide-cytochrome b5 reductases.

Effect of chronic ethanol feeding on the activities and submicrosomal distribution of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase and the demethylases for aminopyrine and ethylmorphine

Effect of chronic ethanol feeding on the activities and submicrosomal distribution of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase and the demethylases for aminopyrine and ethylmorphine