Cortex Microsomes Research Articles

Topology of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4- isomerase in adrenal cortex mitochondria and microsomes

Topology of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4- isomerase in adrenal cortex mitochondria and microsomes

Topology of 3 beta-hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase in adrenal cortex mitochondria and microsomes.

3 beta-Hydroxy-5-ene-steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta HSD) is a NAD(+)-dependent membrane-bound enzyme that catalyzes the oxidation of delta 5-3 beta-hydroxysteroids to delta 4-3-keto structures during adrenal, gonadal, and placental steroidogenesis. Enzyme activity is located in both microsomes and mitochondria. In these experiments we examined the membrane topologies of 3 beta HSD in rat and calf adrenal microsomes and mitochondria by comparing access to the active sites of coenzyme and the inhibitor mersalyl, a nonpenetrant organic mercurial anion. Microsomal activity required exogenous NAD+ and was inhibited by mersalyl, indicating that the active site faced the medium in vitro and the cytoplasm in vivo. In contrast, mitochondrial 3 beta HSD used matrix space NAD+, was inhibited by reduction of intramitochondrial NAD(P)+, and was insensitive to mersalyl. Mitochondrial activity was decreased by exogenous NADH (apparent Ki, 2.8 microM) and increased by added NAD+ (apparent Ka, 2.4 microM). However, mersalyl blocked the effects of exogenous NADH and NAD+ and returned the activity to that observed before coenzyme addition. The membrane-sidedness of the NAD+ activation was examined further in submitochondrial particles prepared by sonication of pyridine nucleotide-depleted calf adrenal cortex mitochondria. Particles were prepared in the absence or presence of 10 mM NAD+ and contained none or 2.9-7.3 nmol NAD+/mg protein, respectively. Both groups of submitochondrial particles required exogenous NAD+ for 3 beta HSD activity, indicating that the active site faced the medium (the particles were everted), and the contained NAD+ was inside the particles. However, 3 beta HSD activity was increased 12-140% in particles that contained NAD+. The results suggest that mitochondrial 3 beta HSD is an integral inner membrane protein, that the active site faces the matrix space and is influenced by coenzyme availability, and that a regulatory site(s) faces the intermembrane space. Binding of NAD+ or NADH to this external site increases or decreases, respectively, the rate of catalysis at the active site. Mitochondrial 3 beta HSD activity may be enhanced by oxidation of intermembrane space NADH via an active rotenone- and antimycin-a-insensitive NADH oxidase.

Effect of spironolactone and phenobarbital administration on bilirubin glucuronidation in hepatic and extrahepatic rat microsomes

We compared bilirubin glucuronidation in liver, renal cortex and intestinal mucosa rat microsomes in order to investigate the effect of individual and combined administration of PB and SP on the formation of bilirubine monoglucuronide (BMG) isomers and bilirubine diglucuronide (BDG)

Metabolism and action of the prostaglandin endoperoxide PGH 2 in rat kidney

Kidney membrane fractions metabolized [1- 14C]PGH 2 to TXB 2, PGE 2, PGF 2α, PGD 2, 6-keto PGF 1α, and HHT. TXA 2, as measured by TXB 2, was enzymatically formed in cortex microsomes and was identified by thin layer chromatography and gas chromatography - mass spectrometry. PGH 2 caused a labile inhibition of cortical PGE 2-stimulated adenylate cyclase. PGE 2, PGF 2α, and PGD 2 are stimulators of cortical adenylate cyclase. The inability of two thromboxane synthetase inhibitors, imidazole and 9,11-azoprosta-5,13 dienoic acid, to block PGH 2 inhibition suggested that TXA 2 was not an obligatory intermediate in this process. Therefore, a potential function of cortical PGH 2 is inhibition of adenylate cyclase.