Regulation of cholesterol biosynthesis in enucleated cells.

In cells that had been physically enucleated after treatment with cytochalasin B (cytoplasts) levels of cholesterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were nearly constant over a 6-h period of time. The ratio of the inactive to the active form of the reductase was unaltered by enucleation and did not change when the cytoplasts were incubated at 37%. The addition to the medium of 25hydroxycholesterol or serum, agents which specifically suppress the reductase activity in nucleated cells, or of cycloheximide, a general inhibitor of protein synthesis, did not affect cholesterol synthesis or reductase activity in the cytoplasts. In contrast compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, rapidly suppressed reductase activity and cholesterol synthesis. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was stable when protein synthesis was blocked by cycloheximide, indicating that degradation of the reductase did not occur in the cytoplasts. Evidence was obtained that other short-lived proteins also were not degraded in the cytoplasts. The stability of 3-hydroxy-3-methylglutaryl-CoA reductase activity levels in cytoplasts appeared, therefore, to be due to the absence of both synthesis and degradation of the reductase. The inability of 25-hydroxycholesterol to suppress reductase activity under these conditions appears to exclude mechanisms involving direct inactivation of the enzyme or indirect inactivation of it through alterations of membrane fluidity or through reversible phosphorylation. These effects of enucleation upon the regulation of sterol synthesis differed from those observed in cells treated with actinomycin D to prevent RNA synthesis. Actinomycin D did not alter the level of 3-hydroxy-3methylglutaryl-CoA reductase, consistent with the effects of enucIeation. But the addition of 25-hydroxycholesterol to actinomycin D-treated cultures resulted in suppression of reductase activity.