Inhibition of p-nitroanisole O-demethylation in perfused rat liver by potassium cyanide

The effect of potassium cyanide on p-nitroanisole O-demethylation in perfused rat livers has been examined. Cyanide (2 mm), an inhibitor of cytochrome oxidase, diminished p-nitroanisole O-demethylation by 50–75% in perfused livers from normal and phenobarbital-treated rats, but had much less effect on hepatic microsomal p-nitroanisole O-demethylation. The inhibition was also observed in livers where the activity of the pentose phosphate shunt was abolished by pretreatment with 6-aminonicotinamide. Cyanide infusion decreased hepatic $\text{ATP}\text{ADP}$ ratios and cellular concentrations of glutamate, α-ketoglutarate, and isocitrate, but caused an increase in the $\text{NADPV}{}^{\mathrm{+}}\text{NADPH}$ ratio. Rates of NADPH generation via the pentose phosphate shunt were unchanged by cyanide, and hepatic concentrations of glucose 6-phosphate were markedly increased by cyanide. Thus, inhibition of p-nitroanisole metabolism could not be explained solely by a direct interaction of cyanide with mixed-function oxidases or diminished NADPH generation via the pentose cycle. These data indicate that cyanide inhibits mixed-function oxidation in intact cells by diminishing the generation of NADPH from sources other than the pentose cycle. Further, these data are consistent with the hypothesis that some NADPH for mixed-function oxidation arises from cyanidesensitive mitochondrial sources.     

Immunohistochemical localization of NADPH-cytochrome c reductase in rat liver.

NADPH-cytochrome $\text{c}$ reductase (NADPH-cytochrome reductase, EC 1.6.2.4), the flavoprotein which is responsible for the NADPH-dependent reduction of cytochromes P-450 in hepatic microsomes, has been localized immunohistochemically at the light microscopic level in rat liver. Localization was achieved through the use of sheep antiserum to rat hepatic microsomal NADPH-cytochrome $\text{c}$ reductase in an unlabeled antibody peroxidase-antiperoxidase technique. Parenchymal cells throughout the liver lobule were found to be stained positively for NADPH-cytochrome $\text{c}$ reductase, although the intensity of immunostaining was slightly greater in the centrilobular regions. Immunostaining for NADPH-cytochrome $\text{c}$ reductase was not detected in Kupffer cells, connective tissue cells, or in cells of the hepatic vasculature.     

Involvement of NADPH-cytochrome c reductase in the rat liver squalene epoxidase system.

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.     

Crystallization of an NADP+-dependent malic enzyme from rat liver

Crystals of a tetrameric NADP+-dependent malic enzyme from rat liver have been grown in the presence of NADP+ using the hanging-drop method of vapour diffusion with ammonium sulphate as the precipitant. Measurement of the crystal density and calculation of the values of Vm for different numbers of polypeptide chains in the unit cell indicate that the asymmetric unit of the crystal contains a complete tetramer, allowing the application of non-crystallographic symmetry to the determination of the molecular structure of this enzyme. This structure would provide only the second example for an enzyme involved in oxidative decarboxylation, the other being 6-phosphogluconate dehydrogenase. In addition, then, to providing an insight into the structure-function relationship in malic enzyme, the successful structure determination would permit valuable comparisons to be made between these two and other enzymes with this catalytic activity.     

The distribution pattern of NADP+-dependent isocitrate dehydrogenase in rat liver

Activities of the NADP+-dependent isocitrate dehydrogenase were measured along the entire sinusoidal path (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the liver of male Wistar rats using a Lowry technique. The measured activities show a slight increase from the periportal to the perivenous end, whereas no such septal-) perivenous gradient could be established. These profiles of enzyme activity give further support to previous studies, suggesting functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed.     

NADPH-cytochrome c (P-450) reductase has the activity of NADPH-linked aquacobalamin reductase in rat liver microsomes.

To elucidate the mammalian system for synthesis of cobalamin coenzymes, microsomal NADPH-linked aquacobalamin reductase was purified and characterized. The enzyme was purified about 534-fold over rat liver microsomal fraction in a yield of about 32%. The purified enzyme was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had a monomeric molecular weight of 79,000. The purified aquacobalamin reductase showed a high specific activity (about 55 mumol/min per mg protein) of NADPH-cytochrome c (P-450) reductase. About 33% of the NADPH-cytochrome c reductase activity found in the microsomal fraction was recovered in the final purified preparation. The activity ratio of NADPH-cytochrome c reductase/NADPH-linked aquacobalamin reductase was about 5.0 through the purification steps, indicating that the rat liver microsomal NADPH-linked aquacobalamin reductase is the NADPH-cytochrome c reductase.     

Regulation of rat liver NADP+-isocitrate dehydrogenase during aging

In an attempt to understand the mechanism of aging in relation to the differences in enzyme regulation, the induction and kinetic properties of NADP⁺ -isocitrate dehydrogenase of the liver of immature (6 weeks), mature (13 weeks), adult (33 weeks) and old (85 weeks) female rats were studied. The specific activity of the cytoplasmic and mitochondrial NADP+ -isocitrate dehydrogenase increased up to the adult age (33 weeks) and decreased in the old rats (85 weeks). Overiectomy decreased and estradiol administration induced activity of both the mitochondrial and eytoplasmic enzyme in the liver ol immature, mature and adult rats but had no significant effect in old rats. However, the activity of mitochondrial NADP⁺ -isocitrate dehydrogenase decreased and eytoplasmic NADP+ -isocitrate dehydrogenase increased following ovariectomy in old rats (85 weeks). Hormone-mediated induction of enzyme activity was actinomycin D sensitive. The Km for isocitrate and NADP, Ki value for oxalomalate, heat stability and electrophoretic mobility of the purified enzyme from the cytosol fraction of the liver of immature and old rats were similar. It can he concluded that the enzyme does not change structurally with age. Part of this work was presented at the 48th Annual General Meeting of the Society of Biological Chemist, India, 1979.     

Immunochemical and immunoelectron microscope studies on localization of NADPH-cytochrome c reductase on rat liver microsomes

By the use of ferritin-conjugated antibody (conjugate) indirect immunoelectron microscopy, NADPH-cytochrome c reductase was localized on rat liver microsomes. Most microsomes in the sections had from 1 to 12 conjugates on their outer surfaces. Among the conjugates, 83% was estimated to bind to NADPH-cytochrome c reductase at a molecular ratio of 1:1, 12% at the ratio of 2:1, and 5% at the ratio of 3 or 4:1. The correlation between immunochemical and morphological data confirmed that most of the NADPH-cytochrome c reducatase reacted with the conjugates. Subsequent morphological analyses have revealed that the enzyme is distributed homogeneously on the outer surfaces of microsomes but heterogeneously within microsomes in groups of three to five enzyme molecules.     

Preparation of homogeneous NADPH-cytochrome P-450 reductase from rat liver.

NADPH-cytochrome P-450 reductase with capacity to support cytochrome P-450-dependent drug metabolism and to reduce artificial electron acceptors has been purified to apparent homogeneity by solubilization with Renex 690 and chromatography on DEAE-Sephadex, Agarose and QAE-Sephadex. The purified protein migrates as a single band on native and SDS-polyacrylamide gel electrophoresis, exhibits a minimum molecular weight of 80,000 daltons and contains 1 molecule each of FAD and FMN per 80,000 molecular weight. The specific activity for cytochrome $\text{c}$ as electron acceptor is 48.8 μmoles per min and for substrate hydroxylation of benzphetamine measured as NADPH oxidation in the presence of cytochrome P-450 and phosphatidylcholine is 2.5 μmoles per min.     

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.     

Inhibition of mixed-function oxidation of p-nitroanisole in perfused rat liver by 2,4-dinitrophenol

The effect of dinitrophenol (52 μm), an uncoupler of oxidative phosphorylation, on p-nitroanisole O-demethylation in the perfused rat liver was examined. Dinitrophenol inhibited p-nitroanisole metabolism 70% in perfused livers from fasted, phenobarbital-treated rats, and 30% in livers from normal rats, but had no effect on this reaction in isolated microsomes. Rates of p-nitroanisole O-demethylation in livers from fed, phenobarbitaltreated rats were not inhibited by dinitrophenol unless the pentose phosphate shunt was first inhibited by 6-aminonicotinamide pretreatment. Dinitrophenol diminished cellular concentrations of ATP and NADPH 30 and 50%, respectively. Since mixed-function oxidation requires NADPH, these data are consistent with the hypothesis that dinitrophenol interrupts the synthesis and/or transfer of reducing equivalents from the mitochondria into the extramitochondrial space by interfering with energy-dependent NADPH synthesis and substrate shuttle mechanisms.In addition, dinitrophenol diminished conjugation reactions 57 and 89% in all metabolic states studied, most likely because it decreased UDP-glucose levels considerably (40 to 60%).     

Alcohol dehydrogenase (ADH) in yeasts. II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica.

In Sm. lipolytica one NAD+-dependent and three NADP+-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD+-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD+-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP+-dependent alcohol dehydrogenases (ADH II--IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP+-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP+-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allylalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.     

Kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP+-dependent dehydrogenases using N6-linked immobilized NADP+ derivatives: Studies with mammalian and microbial glutamate dehydrogenases

This study is concerned with the development and application of kinetic locking-on and auxiliary tactics for bioaffinity purification of NADP(+)-dependent dehydrogenases, specifically (1) the synthesis and characterization of highly substituted N(6)-linked immobilized NADP(+) derivatives using a rapid solid-phase modular approach; (2) the evaluation of the N(6)-linked immobilized NADP(+) derivatives for use with the kinetic locking-on strategy for bioaffinity purification of NADP(+)-dependent dehydrogenases: Model bioaffinity chromatographic studies with glutamate dehydrogenase from bovine liver (GDH with dual cofactor specificity, EC 1.4.1.3) and glutamate dehydrogenase from Candida utilis (GDH which is NADP(+)-specific, EC 1.4.1.4); (3) the selection of an effective "stripping ligand" for NADP(+)-dehydrogenase bioaffinity purifications using N(6)-linked immobilized NADP(+) derivatives in the locking-on mode; and (4) the application of the developed bioaffinity chromatographic system to the purification of C. utilis GDH from a crude cellular extract.Results confirm that the newly developed N(6)-linked immobilized NADP(+) derivatives are suitable for the one-step bioaffinity purification of NADP(+)-dependent GDH provided that they are used in the locking-on mode, steps are taken to inhibit alkaline phosphatase activity in crude cellular extracts, and 2',5'-ADP is used as the stripping ligand during chromatography. The general principles described here are supported by a specific sample enzyme purification; the purification of C. utilis GDH to electrophoretic homogeneity in a single bioaffinity chromatographic step (specific activity, 9.12 micromol/min/mg; purification factor, 83.7; yield 88%). The potential for development of analogous bioaffinity systems for other NADP(+)-dependent dehydrogenases is also discussed.