Studies in vitro on the biosynthesis of ceramide and sphingomyelin. A reevaluation of proposed pathways.

The postulated biosynthetic-pathways of ceramide and sphingomyelin were reinvestigated in extensive investigations by means of synthetic stereo- and radio-chemically pure substrates of high specific radioactivity. As a result, the synthesis of ceramides requires the acyl-CoA-mediated acyltransfer to the long chain bases sphingenine and sphinganine. During the biosynthesis of sphingomyelins, phosphocholine is being transferred from the donor CDP-choline to the primary alcohol group of ceramides. Neither can the free long chain sphingosine bases act as acceptor molecule for the phosphocholine group from CDP-choline, nor has a transfer of [N-14CH3]phosphocholine from [N-14CH3]phosphatidyl choline to ceramide by rat liver enzyme preparations been observed. In agreement with previous studies in vivo, the acylation of sphingenylphosphocholine by acyl-CoA or free fatty acid, ATP and CoASH as an alternative pathway in sphingomyelin biosynthesis has been excluded. Other parameters of the CDP-choline:ceramide cholinephosphotransferase reaction (pH-optimum, ion requirement, competitive inhibition by diacyl glycerols, chain length of fatty acids) are reported. Sphingenine-containing ceramide species are preferred as acceptor molecules. Ceramide species with the L-threo (2S,3S)-enantiomeric long-chain bases are better acceptors than the corresponding D-erythro (2S,3R)-isomeric compounds. The meaning of the steric arrangement for the reaction is discussed.