The mechanism and regulation of dolichyl phosphate biosynthesis in rat liver.

Rat liver slices were pulse labeled for 6 min with [3H]mevalonolactone and then chased for 90 min with unlabeled mevalonolactone in order to study the mechanism of dolichyl phosphate biosynthesis. The cholesterol pathway was also monitored and served to verify the pulse-chase. Under conditions in which radioactivity in the methyl sterol fraction chased to cholesterol, radioactivity in alpha-unsaturated polyprenyl (pyro)-phosphate chased almost exclusively into dolichyl (pyro)phosphate. Lesser amounts of radioactivity appeared in alpha-unsaturated polyprenol and dolichol, and neither exhibited significant decline after 90 min of incubation. The relative rates of cholesterol versus dolichyl phosphate biosynthesis were studied in rat liver under four different nutritional conditions using labeled acetate, while the absolute rates of cholesterol synthesis were determined using 3H2O. From these determinations, the absolute rates of dolichyl phosphate synthesis were calculated. The absolute rates of cholesterol synthesis were found to vary 42-fold while the absolute rates of dolichyl phosphate synthesis were unchanged. To determine the basis for this effect, the rates of synthesis of cholesterol and dolichyl phosphate were quantitated as a function of [3H]mevalonolactone concentration. Plots of nanomoles incorporated into the two lipids were nearly parallel, yielding Km values on the order of 1 mM. In addition, increasing concentrations of mevinolin yielded parallel inhibition of incorporation of [3H]acetate into cholesterol and dolichyl phosphate. The specific activity of squalene synthase in liver microsomes from rats having the highest rate of cholesterol synthesis was only 2-fold greater than in microsomes from rats having the lowest rate. Taken together, the results suggest that the maintenance of constant dolichyl phosphate synthesis under conditions of enhanced cholesterogenesis is not due to saturation of the dolichyl phosphate pathway by either farnesyl pyrophosphate or isopentenyl pyrophosphate but coordinate regulation of hydroxymethylglutaryl-CoA reductase and a reaction on the pathway from farnesyl pyrophosphate to cholesterol.