Selective induction of coumarin 7-hydroxylase by pyrazole in D2 mice

Pyrazole, was given to DBA/2N (D2), C57BL/6N (B6) and AKR/N mice to study its effects on hepatic drug metabolism. A decrease in the total amount of microsomal cytochrome P-450 as well as in the activities of ethylmorphine demethylase and benzo[a]pyrene hydroxylase was found. On the other hand ethoxycoumarin de-ethylase was increased 1.5-2.5-fold (depending on the strain of mouse) and coumarin 7-hydroxylase as much as sevenfold (but only in D2 mice) after pyrazole treatment. This increase was much higher than that caused by phenobarbital, the only well known inducer of coumarin 7-hydroxylase. By reconstituting the mono-oxygenase complex after purification of cytochrome P-450 we found a 40-fold increase in coumarin 7-hydroxylase and eightfold increase in ethoxycoumarin de-ethylase after pyrazole treatment. This was found only in D2 mice. An antibody previously developed against a cytochrome P-450 fraction from the the D2 strain with a high coumarin 7-hydroxylase activity inhibited the microsomal coumarin 7-hydroxylase almost 100% after pyrazole pretreatment of the animals. In the case of control or phenobarbital-treated mice the inhibition was somewhat weaker. With the reconstituted mono-oxygenase complex the inhibition of coumarin 7-hydroxylase was almost 100% both for control and pyrazole-treated D2 mice. The data indicate that pyrazole causes an induction of the microsomal monooxygenase complex different from that caused by phenobarbital or 3-methylcholanthrene and selective for coumarin 7-hydroxylation or 7-ethoxycoumarin de-ethylation. This induction was strong in D2, weak in B6 and absent in AKR/N mice.     

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Hepatic mitochondrial coumarin 7-hydroxylase: comparison with the microsomal enzyme

The specific activity of cytochrome P450-linked coumarin 7-hydroxylase (COH) of hepatic mitoplasts from DBA/2N mice is up to 55% as great as the microsomal activity. According to Western blot and immunodiffusion analysis and inhibition studies with anti-P450Coh and metyrapone, the mitoplastic P450Coh had the same molecular weight and immunochemical and catalytic properties as the corresponding microsomal enzyme. The inducibility of the two proteins by pyrazole and their genetic regulation, as studied with DBA/2N and AKR/J mice, appears to be similar. However, the mitochondrial electron transfer system was not able to support the COH activity of reconstituted microsomal P450Coh although the enzyme was fully active with the microsomal NADPH-cytochrome P450 reductase. This indicates some differences between the two proteins with respect to their interaction with the electron transfer system. This was confirmed by the ability of anti-adrenodoxin reductase antibody to effectively inhibit the mitoplastic COH but not the COH reconstituted with purified microsomal P450Coh and NADPH-P450 reductase. We have previously found that P450Coh does not react with anti-P450b or anti-P450c antibodies, which recognize respective isoforms in rat liver mitoplasts. While P450Coh from microsomes and mitoplasts possess a number of properties in common, the mitoplast P450Coh represents a new subspecies of mitochondrial P450. Some characteristics of mitoplast P450Coh may be the result of post-translational modifications necessary for processing and translocation into the mitochondria.     

A Spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes

1. The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH₂ and oxygen. Activity with NADH₂ was one-third of that with NADPH₂. 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at −12°. Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2′-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.     

Cholesterol 7α-hydroxylase of rat liver: An insulin sensitive enzyme

Four lines of evidence indicates that cholesterol-7α-hydroxylase (ch-7α-H, rate limiting enzyme of cholesterol catabolism) is an insulin sensitive enzyme. 1) Streptozotocin induced diabetes in the rat causes a marked increase in the hepatic activity of ch-7α-H within 24 hrs. with no further increase in subsequent days. 2) Insulin injection can rapidly (within 24 hours) suppress the elevated enzyme activity to normal levels. 3) Insulin $\text{in vitro}$ (0.02 U/ml) can directly suppress ch-7α-H activity in isolated rat liver microsomes or in liver homogenates. 4) Upon exposure to insulin, microsomal ch-7α-H activity showed a reduced stimulatory response to post-microsomal supernatant factors. These studies suggest that a) ch-7α-H is an insulin sensitive enzyme and b) insulin might have a direct role in suppressing ch-7α-H activity in rat liver.     

Posttranscriptional regulation of de novo lipogenesis by glucose-induced O-GlcNAcylation

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