Enzymatic formation of hydroxy ceramides and comparison with enzymes forming nonhydroxy ceramides

Abstract

Radioactive 2-hydroxystearic and cerebronic acids were converted to the coenzyme A thio esters, then tested for reactivity with -sphingosine. Microsomes from mouse brain were found to catalyze the formation of ceramides containing both hydroxy acids. Both the - and -forms of the hydroxy acids reacted. Comparisons of reactivity were made with stearoyl and lignoceroyl CoA, the analogous nonhydroxy acids, which also form ceramides. The ratios of activities of the substrates were found to vary with animal age, with various subcellular fractions, with different rat brain cell preparations, and with different mouse organs. Competition experiments with mixtures of thio ester substrates showed that stearate and lignocerate did not interfere with each other in the formation of ceramide, but hydroxystearoyl CoA inhibited the utilization of the two nonhydroxy substrates and cerebronoyl CoA inhibited the utilization of lignoceroyl CoA. The kinetics of the inhibitions indicated that the effects were noncompetitive. A similar type of inhibition was seen with stearoyl CoA against hydroxystearate incorporation. On the basis of these findings, we suggest that four different enzymes are involved in the acyl transfer reaction: for stearate, hydroxystearate, lignocerate, and cerebronate. Neuronal cell preparations were found to be relatively rich in stearate transferase, while glial cells were relatively rich in hydroxystearate and lignocerate transferases.