Cytochrome P-450scc activity and substrate supply in human placental trophoblasts.

The degree of saturation of cytochrome P-450scc with cholesterol and the substrate turnover number of the cytochrome in cultured trophoblasts and mitochondria from the human placenta were investigated. Cholesterol sulfate was found to be a suitable substrate for probing the degree of saturation of cytochrome P-450scc with substrate during culture and in isolated mitochondria, since it enabled the maximum velocity of the cholesterol side-chain cleavage reaction to be estimated. In contrast, 25-hydroxycholesterol and low density lipoprotein supported trophoblast progesterone production at lower rates than that measured with saturating cholesterol sulfate. In the absence of exogenous substrate, the highest rate of progesterone synthesis by trophoblasts was observed at the beginning of the culture. With cholesterol sulfate as substrate, the turnover number of cytochrome P-450scc in cultured cells was 2.8 min-1 and was not significantly different to the turnover number of the cytochrome for placental mitochondria, where cholesterol is known to be saturating. Results indicate that cholesterol is limiting for progesterone synthesis in cultured trophoblasts even in the presence of lipoprotein rich medium and 8-bromo-cAMP. The concentration of cytochrome P-450scc in trophoblasts was only 20% of that measured for placental homogenate suggesting an induction of the cytochrome occurs when the trophoblasts fuse in vivo to form syncytiotrophoblasts.

Cytochrome P-450scc activity and substrate supply in human placental trophoblasts.

The degree of saturation of cytochrome P-450scc with cholesterol and the substrate turnover number of the cytochrome in cultured trophoblasts and mitochondria from the human placenta were investigated. Cholesterol sulfate was found to be a suitable substrate for probing the degree of saturation of cytochrome P-450scc with substrate during culture and in isolated mitochondria, since it enabled the maximum velocity of the cholesterol side-chain cleavage reaction to be estimated. In contrast, 25-hydroxycholesterol and low density lipoprotein supported trophoblast progesterone production at lower rates than that measured with saturating cholesterol sulfate. In the absence of exogenous substrate, the highest rate of progesterone synthesis by trophoblasts was observed at the beginning of the culture. With cholesterol sulfate as substrate, the turnover number of cytochrome P-450scc in cultured cells was 2.8 min-1 and was not significantly different to the turnover number of the cytochrome for placental mitochondria, where cholesterol is known to be saturating. Results indicate that cholesterol is limiting for progesterone synthesis in cultured trophoblasts even in the presence of lipoprotein rich medium and 8-bromo-cAMP. The concentration of cytochrome P-450scc in trophoblasts was only 20% of that measured for placental homogenate suggesting an induction of the cytochrome occurs when the trophoblasts fuse in vivo to form syncytiotrophoblasts.

Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization.

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.