Pregnenolone

About: Pregnenolone is a research topic. Over the lifetime, 3539 publications have been published within this topic receiving 126444 citations. The topic is also known as: (3b)-3-hydroxy-Pregn-5-en-20-one & 3-Hydroxypregn-5-en-20-one.

Preparation of antiserum to rat cytochrome P-450 cholesterol side chain cleavage, and its use for ultrastructural localization of the immunoreactive enzyme by protein A-gold technique

Abstract: Rabbit antiserum to rat cytochrome P-450 cholesterol side chain cleavage (P-450scc) was produced without a previous biochemical purification of the enzyme. Instead, for immunization we used a single protein band of mol wt 53,000, which was isolated from sodium dodecyl sulfate polyacrylamide gel electrophoresis of rat steroidogenic mitochondrial membranes. The resulting antiserum cross-reacted in a proteinblotting test with affinity purified and biologically active bovine adrenocortical P-450scc enzyme. The antiserum to the rat P-450scc also substantially blocked the conversion of cholesterol to pregnenolone in sonicated steroidogenic mitochondria, suggesting a successful cross-reactivity with the native form of the enzyme, despite the fact that the immunizing antigen was sodium dodecyl sulfate-denatured protein. The antiserum was applied for ultrastructural immunocytochemical visualization of the P-450scc in thin sections of adrenal cortex and immature ovary. Immunoreactive enzyme was identified by the pr...

The Effect of Ether Anaesthesia Stress on Cholesterol‐Side‐Chain Cleavage and Cytochrome P450 in Rat‐Adrenal Mitochondria

Abstract: Cholesterol side-chain cleavage has been studied in intact adrenal mitochondria from rats subjected to stress by ether anaesthesia as a means of raising the plasma adrenocorticotrophin levels. Control rats were either injected with cycloheximide or kept in a quiescent state. After initiation of cholesterol side-chain cleavage by addition of isocitrate, pregnenolone formation from endogenous cholesterol in intact rat-adrenal mitochondria follows a biphasic time-course of formation with an initial rapid phase lasting 3–5 min followed by a much slower rate of formation. Pregnenolone formation from [4-14C]cholesterol is linear if the tracer substrate is added to the mitochondria together with isocitrate. Preincubation of the mitochondria with [4-14C]cholesterol prior to addition of isocitrate results in a biphasic [4-14C]pregnenolone formation; the rate of the initial rapid phase depending on the duration of preincubation. The effect of stress is to increase 2–3 times the rate of pregnenolone formation in the initial phase compared to the rates observed in mitochondria from quiescent or cycloheximide-treated rats. Depletion of cholesterol from adrenal mitochondria follows a similar pattern to pregnenolone formation, but the initial cholesterol content of the mitochondria is not affected by the pretreatment. On the basis of these results it is suggested that only a fraction of the total mitochondrial cholesterol is readily available for cholesterol side-chain cleavage and that this pool is increased by stress. The cytochrome P450 type II spectral changes induced by addition of pregnenolone and isocitrate to the intact rat adrenal mitochondria have also been studied. The effect of stress was to increase pregnenolone-induced difference spectrum in adrenal mitochondrial two- to three-fold compared with cycloheximide-treated animals. Similar results were found for the isocitrate-induced type II difference spectrum. The spectral changes are interpreted to mean that stress causes an increase in the cholesterol complex of side-chain cleavage cytochrome P450. Metabolism of the remainder of the cholesterol in the mitochondria is limited by the rate of transport, or binding, to this reactive centre. It is proposed that the acute effect of stress, mediated by adrenocorticotrophin, is to increase the proportion of mitochondrial cholesterol in the readily available form perhaps by an increase in the rate of transport or binding of cholesterol to sites from which it can be readily metabolised.

The 17, 20-lyase activity of cytochrome P450c17 from human fetal testis favors the Δ5 steroidogenic pathway

Abstract: Cytochrome P450c17 catalyzes both 17alpha-hydroxylation and 17,20-lyase conversion of 21-carbon steroids to 19-carbon precursors of sex steroids. P450c17 can mediate testosterone biosynthesis via the conversion of pregnenolone to dehydroepiandrosterone (the delta(5) pathway) or via conversion of progesterone to androstenedione (the delta(4) pathway). In many species, the 17, 20-lyase activity of P450c17 for one pathway dominates, reflecting the preferred steroidogenic pathway of that species. All studies of recombinant human P450c17 and of human adrenal microsomes have found high 17, 20-lyase activity only in the delta(5) pathway. Because the 17, 20-lyase activities in both the delta(4) and delta(5) pathways for testicular P450c17 have not been directly compared, however, it is not known if the delta(5) pathway dominates in the human testis. To resolve this issue, we assayed the conversion of 17alpha-hydroxypregnenolone to dehydroepiandrosterone (delta(5) 17, 20-lyase activity) and of 17alpha-hydroxyprogesterone to androstenedione (delta(4) 17, 20-lyase activity) by human fetal testicular microsomes. We obtained apparent Michaelis constant (K(m)) and maximum velocity (V(max)) values of 1.0 microM and 0.73 pmol.min(-1). microg(-1) for delta(5) 17, 20-lyase activity and of 3.5 microM and 0.23 pmol.min(-1). microg(-1) for delta(4) 17, 20-lyase activity. Catalytic efficiencies, expressed as the ratio V(max)/K(m), were 0.73 and 0.066 for the delta(5) and delta(4) reactions, respectively, indicating 11-fold higher preference for the delta(5) pathway. We conclude that the majority of testosterone biosynthesis in the human testis proceeds through the conversion of pregnenolone to dehydroepiandrosterone via the delta(5) pathway.