scholarly journals Ring-and N-hydroxylation of 2-acetamidofluorene by rat liver reconstituted cytochrome P-450 enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 561-564 ◽  
Author(s):  
P D Lotlikar ◽  
K Zaleski
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Enzyme System ◽  
Partial Purification ◽  
Hydroxylated Metabolites ◽  
Triton X ◽  
Cytochrome P 450 ◽  
Ring Hydroxylation ◽  
Hydroxylation Activity ◽  
Nadph Cytochrome C Reductase

The N- and ring-hydroxylation of 2-acetamidofluorene were studied with a reconstituted cytochrome P-450 enzyme from microsomal fractions of liver from both control and 3-methylcholanthrene-pretreated rats. Proteinase treatment and Triton X-100 solubilization were two important steps for partial purification of the cytochrome P-450 fraction. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions were required for optimum N- and ring-hydroxylation activity. Hydroxylation activity was determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from pretreated animals was severalfold more active than the fraction from controls. Formation of N-hydroxylated metabolites with reconstituted systems from both control and pretreated animals was greater than that with their respective whole microsomal fractions.

scholarly journals Dimethylnitrosamine demethylation by reconstituted liver microsomal cytochrome P-450 enzyme system

1975 ◽  
Vol 152 (3) ◽  
pp. 705-708 ◽  
Author(s):  
P D Lotlikar ◽  
W J Baldy ◽  
E N Dwyer
Keyword(s):  
Cytochrome C ◽  
Enzyme System ◽  
Microsomal Cytochrome ◽  
Cytochrome C Reductase ◽  
Oxidative Demethylation ◽  
Enzyme Systems ◽  
Triton X ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase ◽  
Demethylation Activity

Oxidative demethylation of dimethylnitosamine was studied with both reconstituted and unresolved liver microsomal cytochrome P-450 enzyme systems from rats and hamsters. Proteinase treatment of liver microsomal preparations yielded cytochrome P-450 particulate fractions. Both cytochrome P-450 and NADPH- cytochrome c reductase fractions were required for optimum demethylation activity. Particulate cytochrome P-450 fractions were more effecient than either Triton X-100- or cholatesolubilized preparations of these particles in demethylation activity with rat and hamster liver preparations appear to be due to differences in specificity in their cytochrome P-450 fractions.

scholarly journals GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES

1973 ◽  
Vol 59 (1) ◽  
pp. 73-88 ◽  
Author(s):  
J. J. M. Bergeron ◽  
J. H. Ehrenreich ◽  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Companion Paper ◽  
Transferase Activity ◽  
Ethanol Treatment ◽  
Liver Homogenates ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

The three Golgi fractions isolated from rat liver homogenates by the procedure given in the companion paper account for 6–7% of the protein of the total microsomal fraction used as starting preparation. The lightest, most homogeneous Golgi fraction (GF1) lacks typical "microsomal" activities, e.g., glucose-6-phosphatase, NADPH-cytochrome c-reductase, and cytochrome P-450. The heaviest, most heterogeneous fraction (GF3) is contaminated by endoplasmic reticulum membranes to the extent of ∼15% of its protein. The three fractions taken together account for nearly all the UDP-galactose: N-acetyl-glucosamine galactosyltransferase of the parent microsomal fraction, and for ∼70% of the activity of the original homogenate. Omission of the ethanol treatment of the animals reduces the recovery by half. The transferase activity is associated with the membranes of the Golgi elements, not with their content. Galactose is transferred not only to N-acetyl-glucosamine but also to an unidentified lipid-soluble component.

scholarly journals Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants

1989 ◽  
Vol 259 (3) ◽  
pp. 847-853 ◽  
Author(s):  
I Benveniste ◽  
A Lesot ◽  
M P Hasenfratz ◽  
F Durst
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Higher Plants ◽  
Polyclonal Antibodies ◽  
Reductase Activity ◽  
Jerusalem Artichoke ◽  
Cross Reactivity ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

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.

scholarly journals Re-examination of the subcellular localization of thyroxine 5′-deiodination in rat liver

1979 ◽  
Vol 180 (2) ◽  
pp. 273-279 ◽  
Author(s):  
D Auf Dem Brinke ◽  
R D Hesch ◽  
J Köhrle
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Rat Liver ◽  
Marker Enzyme ◽  
Cytochrome C Reductase ◽  
Substrate Saturation ◽  
Single Enzyme ◽  
Optimal Ph ◽  
Nadph Cytochrome C Reductase

We describe the existence of at least two thyroxine 5′-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5′-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5′-deiodination. However, this process is not mediated by only a single enzyme.

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