Biochemical characterization of the reverse activity of rat brain ceramidase - A CoA-independent and fumonisin B-1-insensitive ceramide synthase

We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue, We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain i nsight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited i n vitro and in cells by fumonisin B-1, an inhibitor of the CoA-dependent ce ramide synthase, The enzyme showed a narrow pH optimum in the neutral range , and there was very low activity in the alkaline range. Substrate specific ity studies were performed, and the enzyme showed the highest activity with D-erythro-sphingosine (K-m of 0.16 mol %, and V-max of 0.3 mu mol/min/mg), but D-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was al so studied, and the highest activity was observed for myristic acid with a K-m of 1.7 mol % and a V-max of 0.63 mu mol/min/mg, Kinetic studies were pe rformed to investigate the mechanism of the reaction, and Lineweaver-Burk p lots indicated a sequential mechanism. Two competitive inhibitors of the tw o substrates were identified, L-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequentia l mechanism. The effect of lipids were also tested. Most of these lipids sh owed moderate inhibition, whereas the effects of phosphatidic acid and card iolipin were more potent with total inhibition at around 2.5-5 mol %, Parad oxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are d iscussed in view of a possible regulation of this enzyme by the intracellul ar pH or by an interaction with cardiolipin and/or phosphatidic acid.