INTERACTION OF SPHINGOMYELINASE WITH SPHINGOMYELIN ANALOGS MODIFIED AT THE C-1 AND C-3 POSITIONS OF THE SPHINGOSINE BACKBONE

In this study, analogs differing at the C-1 or C-3 position of the sph ingosine backbone of sphingomyelin were examined in neutral pH-optimum sphingomyelinase assays. Two analogs modified at the C-1 position, ce ramide-1-phosphate and ceramide-1-phosphoethanol-N,N-dimethylamine, co uld act as modest substrates but showed no ability to inhibit the reac tion when egg sphingomyelin was used as the substrate. Four analogs of sphingomyelin differing at the C-3 position were used in which the hy droxyl group was replaced by a hydrogen atom (to give a deoxy-sphingom yelin analog), or with a O-methyl, O-ethyl or O-tetrahydropyranyl grou p. The deoxy analog showed no ability to compete with substrate of sph ingomyelinase nor could it be hydrolyzed by the enzyme, suggesting tha t the hydroxyl group is a required substituent for the substrate. The 3-O-methyl and 3-O-ethyl-sphingomyelin analogs were inhibitors, with I C50 values of 50 mu M and 140 mu M, respectively, at standard assay co nditions. However, when the rat brain acidic pH-optimum sphingomyelina se was used, no inhibition by the 3-O-methyl analog could be detected. The size of the alkyl group on the ether moiety was important, as sho wn by the inability of 3-O-tetrahydropyranyl-sphingomyelin to compete with substrate of neutral pH-optimum sphingomyelinase.