Estrogen synthetase in the horse. Comparison of equine placental and rat liver NADPH-cytrochrome c (P-450) reductase activities.

NADPH-cytochrome c (P-450) reductases from horse placenta and rat liver were purified and their biological activities compared using cytochrome c as substrate. Rat liver reductase was purified to electrophoretic homogeneity in one chromatographic step on 2',5'-ADP agarose, and had a relative mass of 85,000 Da as estimated by SDS-PAGE. Equine placental reductase was separated from cytochrome P-450 on aminohexyl-Sepharose 4B and further purified on 2',5-ADP agarose; this preparation exhibited two bands, one of 85,000 and one of 80,000 Da, on SDS-PAGE. The lower molecular weight form was assumed to be a proteolytic product of the higher molecular weight form. A high retention of activity was obtained in both preparations. Equine placenta and rat liver enzymes were found to exhibit very similar Vmax and Km, suggesting that they are not species specific.

Characterization of lipoprotein lipase activators from equine plasma.

Equine plasma lipoproteins were fractionated into VLDL, LDL-1, LDL-2 and HDL by density gradient ultracentrifugation. From each lipoprotein fraction, five apo C like peptides of approx. M(r) 1400, 10000, 9500, 9000 and 8000 were detected by SDS-polyacrylamide gel electrophoresis. After partial purification by Sephadex G-75, one fraction, showing a strong activation of lipoprotein lipase, was further purified by Mono Q anion exchange column. Two of the apo C like peptides (M(r) 10000 and 8000) activated the bovine milk lipoprotein lipase in vitro; only one (M(r) 9500) inhibited the lipolytic activity. This work confirms that many mammals present two apo C-II components with different molecular weights.

Transfer of cholesterol between high density lipoproteins and cultured rat Sertoli cells.

In the testes, the Sertoli cells are separated from the blood capillaries by the basement membrane, thereby excluding the passage of low density lipoproteins (LDLs) but allowing the passage of high density lipoproteins (HDLs). The present study examines first the capacity of Sertoli cells to uptake cholesterol from HDL and secondly the role of apolipoproteins (apo) A-I and E in cholesterol flux between HDL and cultured rat Sertoli cells. In the presence of HDL in cultured medium, rat Sertoli cells accumulated few amounts of esterified cholesterol. Incubation of [14C] cholesterol-labelled Sertoli cells with [3H]cholesterol-labelled HDL showed that the amount of cholesterol influx slightly exceeded its efflux, thus resulting in a net uptake of cholesterol from HDL to rat Sertoli cells. The amount of HDL-cholesterol converted to steroids by Sertoli cells was about 32% of influx. Uptake of cholesterol by Sertoli cells was three times higher with phospholipid-apo A-I vesicles and seven times higher with phospholipid- apo E vesicles than that with phospholipid vesicles without apolipoprotein. Phospholipid- apo A-I vesicles promoted cholesterol efflux at the same rate as native HDL and twice as efficiently as phospholipid- apo E vesicles. Thus, this study shows that rat Sertoli cells have the capacity to take up HDL-cholesterol for membrane renewal and steroid production mainly by apo E dependent pathways.

Effect of lipoproteins on cholesterol synthesis in rat Sertoli cells.

Lipoprotein metabolism has been investigated in cultured rat Sertoli cells. Cells incubated with low-density lipoproteins (LDLs) or high-density lipoproteins (HDLs) showed a concentration-dependent decrease of sterol synthesis, indicating a net cholesterol delivery to the Sertoli cells. At 50 micrograms/mL, lipoproteins inhibited the incorporation of [14C]acetate into free cholesterol by 83% for the LDL and 47% for the HDL. Electron microscopic examinations of the Sertoli cells provide evidence of the internalization of gold-labelled HDL into coated pits and coated vesicles. Competitive studies between human LDL and rat HDL indicate that Sertoli cells take up cholesterol from LDL and HDL containing apolipoprotein (apo) E by common pathways. These results suggest that Sertoli cells possess apo B and E receptors for the uptake and degradation of LDL and HDL, although the basement membrane excludes the passage of LDL from blood capillaries to the Sertoli cells. At 50 micrograms/mL, apo-E-depleted HDL inhibited the incorporation of [14C]acetate into free cholesterol by 34%. Thus, this study shows that Sertoli cells are capable of taking up apo-E-depleted HDL cholesterol for cell metabolism.