Abstract
We have followed the transfer of newly synthesized cholesterol to the plasma membrane in cultured fibroblasts using cholesterol oxidase as a probe. Since the enzyme has access only to the plasma membrane in intact cells, it permits the discrimination of cell surface and endogenous cholesterol. Cholesterol synthesized from radiolabeled acetate was transferred to the plasma membrane in a strictly first order fashion with a half-time of 1-2 h at 37 degrees C. The rate of transfer was similar in rapidly growing and confluent cells and was not affected by preincubating the cells in lipoprotein-deficient serum which greatly stimulated cholesterol synthesis. We used equilibrium density gradient centrifugation of homogenates from cholesterol oxidase-treated cells to examine further the distribution of newly synthesized cholesterol between cellular pools. We identified membrane fractions enriched in newly synthesized cholesterol yet inaccessible to cholesterol oxidase. The cholesterol in these membranes eventually moved to the plasma membrane. The movement of exogenous radiocholesterol from the plasma membrane to the cell interior also was examined by this method. No detectable transfer was observed over several hours, during which time endogenous cholesterol moved to the plasma membrane. We conclude that the transfer of newly synthesized cholesterol to the plasma membrane is a vectorial process and is not mediated by a simple diffusional equilibrium.
Highlights
We recently have developed a technique which permits intact cells, it permits the discriminatioonf cell surface plasma membrane and endogenous cholesterol pools to be andendogenouscholesterol.Cholesterolsynthesized distinguished in intact cells [5]
In agreementwith previous reports [14], we found that the incorporation of radiolabeled acetate into cholesterol was dramaticallyenhanced if cells were preincubated in a medium containing lipoprotein-deficient serum
Incorporation into theplasma membrane initially lagged behind total synthesis (Fig. 1)but after about 2 h the rate of appearance of radiocholesterol atthe cell surface became parallel to the rateof synthesis
Summary
Time Course of Incorporation of PHIAcetate into Cellular Cholesterol-Human foreskin fibroblasts synthesize radiocholesterol from labeled acetate. This corresponds to a half-time of 0.86 h for the transfer of newly synthesized cholesterol to theplasma membrane in this experiment. This method had the advantage that variations between flasks in the incorporation of [3H]acetateinto cholesterol did not affect the data since the parameter fit was the fraction susceptible to oxidation
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