Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes

Abstract

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.