John L. Purvis

Lifetime of microsomal cytochrome P-450 and steroidogenic enzymes in rat testis as influenced by human chorionic gonadotrophin

The lifetime of different microsomal steroidogenic enzymes and the cytochrome components of the NADPH-cytochrome P-450 pathway have been determined in rat testis by measuring their decrease logarithmically after hypophysectomy. Although both cytochrome P-450 and 17α-hydroxylase show biphasic decay curves, the first decay curve contains 89–94% of the cytochrome P-450 and 17α-hydroxylase levels. Steroidogenic enzymes which are located mainly in the leydig cells, decay much faster than microsomal protein, t12 = 12 days, which represents mainly decay of tubular protein. The similarity between the major half-life of cytochrome P-450, t12 = 3.3 days, 17α-hydroxylase, t12 = 2.3 days and the C17–C20 lyase, t12 = 3.4 days and the uniformity of their response to human chorionic gonadotrophin (HCG) provides additional evidence that these two steroidogenic enzymes require cytochrome P-450. Both the 17α-hydroxylase and the C17–C20 lyase were shown to have a constant activity per nmole of cytochrome P-450 during a sixfold change in the level of cytochrome P-450 brought about by HCG treatment of rats with intact pituitaries. The decay of 17β-hydroxysteroid dehydrogenase, t12 = 4.5 days, was slower than P-450 dependent enzymes. Rats with intact pituitaries are not under maximal stimulation by endogenous LH because addition of HCG increases the levels of microsomal and mitochondrial cytochrome P-450 220 and 1620%, respectively. The rates of synthesis during the increase from one cytochrome P-450 level to another was calculated at 0.1182 testes/day for microsomal cytochrome P-450 and 0.10 nmoles/2 testes/day for mitochondrial cytochrome P-450. Treatment of hypophysectomized rats with HCG results in large increases of cytochrome P-450, 17α-hydroxylase, C17–C20 lyase and 5α-reductase, but not cytochrome b5, microsomal protein, 7α-hydroxylase, or the 17β-hydroxysteroid dehydrogenase. While it is clear that the two cytochrome P-450 dependent hydroxylases involved in steroidogenesis and the 5α-reductase are under the control of gonadotrophin, it is not clear how 17β-hydroxysteroid dehydrogenase levels are maintained or in what manner the 5α-reductase level is controlled in mature animals.

Studies of cytochrome P-450 in testis microsomes

Abstract Studies on the role of cytochrome P-450 in mouse, rat, and chick testis microsomes showed that this CO-binding hemoprotein is involved in the activity of the 17α-hydroxylase. A 70–80% inhibition by CO of the 17α-hydroxylase activity was detected in rat and chick testis microsomes. In the mouse testis, the level of the enzyme activity is ten times greater than that of the rat. This partly explains why an acceleration of NADPH oxidation by progesterone can be observed in mouse but not in rat testis microsomes. In rat testis microsomes, type I binding spectra of cytochrome P-450 was observed with pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. The apparent K s values for progesterone and 17-hydroxyprogesterone were 0.50 and 1.00 μ m , respectively. When NADPH is used to measure cytochrome P-450 levels in rat testis microsomes, CO formation resulting from a stimulation in lipid peroxidation by phosphate or Fe 2+ was sufficient to bind with 50% of the total amount of cytochrome P-450. Substitution of phosphate by Tris reduced the amount of lipid peroxidation to minimal levels. On a comparable basis, no CO formation was observed in avian testis microsomes. An increase in the testicular levels of cytochrome P-450 resulted upon the administration of HCG and cyclic-AMP to 1-day-old chicks. The lack of stimulation of the cytochrome P-450 levels by progesterone and pregnenolone suggest that the hormonal stimulation of the P-450 levels is not due to substrate induction.

Control of cytochrome P-450 in rat testis mitochondria by human chorionic gonadotrophin

Cytochrome P-450 cannot be detected spectrophotometrically in testis mitochondria of untreated rats because of the high cytochrome a3 to Cytochrome P-450 ratio. Injection of human chorionic gonadotrophin (HCG) causes a large increase in mitochondrial cytochrome P-450. After 14 days injection, mitochondrial cytochrome P-450 levels are increased 15- to 30-fold (from 0.007 to 0.134 nmoles/mg protein) over control levels. Levels of cytochrome a + a3 are not altered by this treatment. Mitochondrial cytochrome P-450 can also be demonstrated by injection of HCG into rats which were hypophysectomized 24 days previously. During hypophysectorny the mitochondrial cytochromes c + ci, a + a3 and mitochondrial protein decay with halflives of 14, 16, and 15.5 days, respectively. HCG treatment for 8 days increases mitochondrial cytochrome P-450 (from < 0.003 to 0.24 nmoles/mg protein) without altering the levels of the other mitochondrial cytochromes. The control of cytochrome P-450 levels in the mitochondria by HCG suggests that the level of this key component of cholesterol side-chain cleavage enzyme may be of importance in the regulation of steroidogenesis in the testis.

The maintenance of mitochondrial size in the rat adrenal cortex zona fasciculata by ACTH

Abstract An electron microscopic examination of the rat adrenal cortex reveals changes in the abundance and structure of the mitochondria of the zona fasciculata after hypophysectomy, which are time-dependent and lead to the production of giant (diameter > 1.5μ) mitochondria. These changes were measured in low power fields involving a total thin section area of 850μ2 at 4, 7, 11, and 14 days after hypophysectomy. Following hypophysectomy, the number of mitochondria decreases, the volume of lipid droplets increases, the ratio of the surface area of the outer mitochondrial membrane to the mitochondrial volume decreases (indicative of increased size), and the percent of small mitochondria increases. The volume of giant mitochondria, comprising 8% of the total mitochondrial volume in control animals, increases to 65% of the total 7 days after hypophysectomy and to 82% 14 days after hypophysectomy. It has been found that administration of ACTH causes the gradual reversal of these effects of hypophysectomy over a period of 9 days. Mitochondrial partitions, clearly associated with the inner membrane, were observed after hypophysectomy and during ACTH-induced recovery. The evidence suggests that ACTH controls the process of growth and division of the mitochondria. The retarded growth of mitochondria in the absence of ACTH is associated with fusion of existing mitochondria to form giant mitochondria.

ENERGY-LINKED INCORPORATION OF DIPHOSPHOPYRIDINE NUCLEOTIDE INTO RAT-LIVER MITOCHONDRIA.

DPN+ is incorporated intact into isolated rat liver mitochondria by an energy-linked process. No difference in incorporation could be seen when DPNe labeled in either the nicotinamide or the adenine portion of the molecule was used. Incorporation is not dependent on endogenous phosphate and proceeds equally well in a State 4 or a State 3 system. The uptake of DPN+ by mitochondria is also stimulated by oligomycin and can be supported by ATP in the presence of antimycin A. This latter reaction is inhibited by both oligomycin and 2,4-dinitrophenol.

Pyridine nucleotide biosynthesis in Claviceps

Abstract Claviceps purpurea grown on synthetic medium incorporated labeled [7- 14 ]nicotinic acid and [7- 14 C]nicotinamide into NaMN, des-NAD, NAD, and NADP. Label also appeared in NMN and N -methyl nicotinamide. The specific activities of NAD, NADP, and NMN are compatible with the operation of the Preiss-Handler pathway of NAD biosynthesis (nicotinic acid → NaMN → des-NAD → NAD → NADP). The relative amounts of NaMN:des-NAD:NAD and NADP were about 8:1:36:10 on incubation of Claviceps with nicotinic acid for 6 hr. The incorporation of nicotinamide into NAD proceeds mainly by conversion to nicotinic acid catalyzed by nicotinamide deamidase. Tryptophan ([U- 14 C]benzene ring) was incorporated into NAD demonstrating the presence of the tryptophan-nicotinic acid pathway. No qualitative difference in pyridine nucleotide intermediates was noted in C. purpurea CPM, which does not produce clavine alkaloids, and Claviceps 47A which does produce clavine alkaloids.