Abstract
Fatty acyl-Co A: sphingosine acyltransferase (ceramide synthase, EC 2.3.1.24) is mainly localized in the microsomal and mitochondrial membranes. Attempts to isolate the enzyme have failed, largely because there has been little or no detection of the enzyme activity in detergent extracts. In this study, we solubilized the membrane-bound enzyme from bovine brain mitochondria with a Tris-HCl buffer containing 2% Triton X-100 and, after removal of the detergent, reconstituted it with the membrane lipid liposomes. The specific activity of the reconstituted enzyme was approx. 8 times higher than that of the solubilized enzyme. We next examined the lipid dependence of the enzyme, using various phospholipid liposomes. The ability of phospholipids to enhance the activity of solubilized ceramide synthase was specific and structure-related. The most potent stimulator was phosphatidylserine liposomes, suggesting an important role of the net negative charges. This paper also describes a highly reproducible high-performance liquid chromatographic (HPLC) procedure for the determination of ceramide synthase activity. Combination of the HPLC method with the reconstituted enzyme system appears to be suitable for elucidating the characteristics of this enzyme.
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