Intermembrane transfer of 5 alpha-cholest-7-en-3 beta-ol. Facilitation by supernatant protein (SCP).

The dehydrogenation of 5 alpha-cholest-7-en-3 beta-ol (lathosterol) to cholest-5,7-dien-3 beta-ol (7-dehydrocholesterol) was studied in rat liver microsomes with [3H]lathosterol as substrate. Microsomal delta 5-dehydrogenase activity was stimulated by purified "squalene and sterol carrier protein" (SCP) (Dempsey, M. E., McCoy, K. E., Barker, H. N., Vafiadou, A. D., Lorsbach, T., and Haward, J. B. (1981) J. Biol. Chem. 256, 1867-1873). When the integrity of microsomal membranes was perturbed by treatment with detergents such as 0.2% Triton X-100 or 0.05% sodium deoxycholate or by phospholipase A2, the stimulatory effect of SCP was abolished, indicating that an intact membrane system is required for the response to SCP. Dehydrogenase solubilized with 2% Triton X-100 was not stimulated by SCP. Lathosterol was effectively incorporated into microsomes in the presence or absence of SCP. The effect of SCP on the enzymatic dehydrogenation of lathosterol previously incorporated into microsomes was significantly greater than on the conversion of exogenous substrate. Kinetically, the effect of SCP was not on initial velocity except to maintain it for much longer periods of time. Tryptic digestion of previously lathosterol-loaded microsomes inactivated the delta 5-dehydrogenase. When such trypsin-treated microsomes containing [3H]lathosterol (donor microsomes) were incubated with normal, enzymatically active microsomes (acceptor microsomes), formation of 7-dehydrocholesterol could not be detected. However, if SCP was included in this assay system, dehydrogenation occurred rapidly, suggesting SCP-mediated intermembrane translocation of lathosterol. When the membranes of acceptor microsomes were damaged by detergent or phospholipase A2, the SCP effect disappeared. SCP-mediated lathosterol transfer was shown more directly by sucrose density gradient centrifugation to separate trypsinized and normal microsomes. These findings show that cytoplasmic proteins promote not only intermembrane transfer of squalene (Friedlander, E. J., Caras, T. W., Liu, L.-F. H., and Bloch, K. (1980) J. Biol. Chem. 255, 8042-8045) but also of later intermediates in cholesterol biosynthesis.