Effect of phenobarbital induction, charcoal treatment and storage on the spectral binding characteristics and NADPH-cytochrome P-450 reductase activity of hepatic microsomes

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.

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Presence of nadph-cytochrome P-450 reductase in rat liver golgi membranes: Evidence obtained by immunoadsorption method

The Journal of Cell Biology ◽

10.1083/jcb.79.2.590 ◽

1978 ◽

Vol 79 (2) ◽

pp. 590-597 ◽

Cited By ~ 55

Author(s):

A Ito ◽

GE Palade

Keyword(s):

Rat Liver ◽

Reductase Activity ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Nadph Cytochrome ◽

Golgi Membranes ◽

Cyt C ◽

Bona Fide ◽

Liver Homogenates ◽

Cytochrome P 450

Light Golgi fractions (GF(1+2)) prepared from rat liver homogenates by a modification of the Ehrenreich et al. procedure (J. Cell Biol. 59:45) had significant NADPH-cytochrome P(450) reductase (NADPH-cyt c reductase) activity if assayed immediately after their isolation. An antibody raised in rabbits against purified microsomal and Golgi fractions. To find out whether this activity is located in bona fide Golgi elements or in contaminating microsomal vesicles, we used the following 3-step immunoadsorption procedure: (a) antirabbit IgG (raised in goats) was conjugated to small (2-5 μm) polycrylamide (PA) beads; (b) rabbit anti NADPH-cyt c reductase was immunoadsorbed to the antibody-coated beads; and (c) GF(1+2) was reacted with the beads carrying the two successive layers of antibodies. The beads were then recovered by centrifugation, and were washed, fixed, embedded in agarose, and processed for transmission electromicroscopy. Antireductase- coated beads absorbed 60 percent of the NADPH-cyt c reductase (and comparable fractions of NADH-cyt c reductase and glucose-6-phosphatase) but only 20 percent of the galactosyltransferase activity of the input GF(1+2). Differential vesicle counts showed that approximately 72 percent of the immunoadsorbed vesicles were morphologically recognizable Golgi elements (vesicles with very low density lipoprotein [VLDL] clusters or Golgi cisternae); vesicles with single VLDL and smooth surfaced microsome-like vesicles were too few (approximately 25 percent) to account for the activity. It is concluded that NADPH-cytochrome P(450) reductase is a Golgi membrane enzyme of probably uneven distribution among the elements of the Golgi complex.

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Complete amino acid sequence of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes

Biochemistry ◽

10.1021/bi00372a018 ◽

1986 ◽

Vol 25 (24) ◽

pp. 7906-7911 ◽

Cited By ~ 52

Author(s):

Mitsuru Haniu ◽

Takashi Iyanagi ◽

Philip Miller ◽

Terry D. Lee ◽

John E. Shively

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Complete Amino Acid Sequence ◽

Nadph Cytochrome ◽

Hepatic Microsomes ◽

Cytochrome P 450

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Temperature Influence on Basal Activity and Induction of Mixed Function Oxygenase Activity in Fundulus heteroclitus

Journal of the Fisheries Research Board of Canada ◽

10.1139/f79-200 ◽

1979 ◽

Vol 36 (11) ◽

pp. 1400-1405 ◽

Cited By ~ 66

Author(s):

John J. Stegeman

Keyword(s):

Cytochrome C ◽

Fundulus Heteroclitus ◽

Reductase Activity ◽

Control Fish ◽

Cytochrome C Reductase ◽

Nadph Cytochrome ◽

Pyrene Hydroxylase ◽

Mixed Function Oxygenase ◽

Cytochrome P 450 ◽

Nadph Cytochrome C Reductase

Treatment of Fundulus heteroclitus acclimated to 6.5 °C with benzo(a)pyrene did not elicit any change in the levels of hepatic microsomal NADH- or NADPH-cytochrome c reductase activity, nor in the levels of cytochrome P-450 or its catalytic activities. However, the same treatment offish at 16 5 °C resulted in a marked induction of benzo(a)pyrene hydroxylase and NADPH-cytochrome c reductase. Cytochrome P-450 content was also higher in the warm, treated fish and the Soret maximum of reduced, CO-treated microsomes was shifted to the violet. Levels of aminopyrine demethylase and NADH-cytochrome c reductase activities did not show a significant treatment effect. At neither temperature could treated and control fish be distinguished on the basis of in vitro inhibition of benzo(a)pyrene hydroxylase activity by 7,8-benzoflavone. Levels of NADPH-cytochrome c reductase and benzo(a)pyrene hydroxylase activities were greater in control Fundulus acclimated to 6.5 °C than to 16.5 °C, when normalized to microsomal protein, but not when based on body weight. The results indicate that habitat temperature alone may not affect the capacity for initial hydrocarbon metabolism in fish, but that it can strongly influence the induction of cytochrome P-450. Key words: temperature, cytochrome P-450, hydrocarbon metabolism, mixed-function oxygenase, Fundulus heteroclitus

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The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells

Biochemical Journal ◽

10.1042/bj1100407 ◽

1968 ◽

Vol 110 (3) ◽

pp. 407-412 ◽

Cited By ~ 145

Author(s):

J. L. Holtzman ◽

T. E. Gram ◽

P. L. Gigon ◽

J. R. Gillette

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Endoplasmic Reticulum ◽

Reductase Activity ◽

Mixed Function Oxidase ◽

Protein Ratio ◽

Nadph Cytochrome ◽

Rate Limiting Step ◽

Significant Difference ◽

The Difference ◽

Cytochrome P 450

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3–5), per μg. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1·7) or per unit of NADPH–cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH–cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.

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Hepatic Microsomal Monooxygenase Activity and the Biotransformation of Hydrocarbons in Deep Benthic Fish from the Western North Atlantic

Canadian Journal of Fisheries and Aquatic Sciences ◽

10.1139/f83-313 ◽

1983 ◽

Vol 40 (S2) ◽

pp. s78-s85 ◽

Cited By ~ 11

Author(s):

John J. Stegeman

Keyword(s):

Cytochrome C ◽

North Atlantic ◽

Deep Sea ◽

Reductase Activity ◽

Environmental Chemicals ◽

Specific Content ◽

Monooxygenase Activity ◽

Hepatic Microsomes ◽

Pyrene Hydroxylase ◽

Cytochrome P 450

Microsomal electron transfer components cytochrome P-450, cytochrome b5, NADPH-cytochrome c (P-450) reductase activity and NADH-cytochrome c (b5) reductase activity were present in hepatic microsomes from several species of deep-sea fishes sampled between 1400 and 3200 m deep in the North Atlantic. The specific content of cytochrome P-450 varied among the species but was within the range observed for shallow-water fishes. The specific content was the highest in the deepest sampled species, Coryphaenoides armatus (0.25 nmol/mg microsomal protein). Hepatic microsomes from this species also exhibited little or no putative cytochrome P-420 whose presence suggested some degradation of cytochrome P-450 in the other species. Ethoxyresorufin O-deethylase and benzo[a]pyrene hydroxylase activities were high in several species, particularly Coryphaenoides armatus. Strong inhibition of benzo[a]pyrene hydroxylase activity by α-naphthoflavone was demonstrated in some species. The data suggest that some hepatic cytochrome P-450 in Coryphaenoides armatus (and possibly Coryphaenoides leptolepis and Bathysaurus mollis) but not in Coryphaenoides rupestris or Antimora rostrata may have been induced by environmental chemicals in the deep sea. As in other teleost fishes, Coryphaenoides hepatic microsomes metabolized benzo[a]pyrene in vitro with a high specificity for the benzo-ring, suggesting the capacity to form mutagenic derivatives of some hydrocarbons efficiently.

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