Neutral magnesium-dependent sphingomyelinase from liver plasma membrane: Purification and inhibition by ubiquinol

Plasma membranes isolated from pig liver contained almost no acid sphingomy elinase but significant neutral magnesium-dependent sphingomyelinase that w as activated by phosphatidylserine. We report here the purification to appa rent homogeneity of neutral sphingomyelinase of about 87 kDa from liver pla sma membranes. The purified enzyme. strictly required magnesium and had a n eutral optimal pH. In contrast with neutral sphingomyelinase purified from other sources (such as brain), the enzyme purified from from liver plasma m embrane was not inhibited by GSH and, strikingly, it was not activated by p hosphatidylserine. Liver sphingomyelinase was inhibited by several lipophil ic antioxidants in a dose-dependent way. Ubiquinol-10 was more effective th an alpha -tocopherol, alpha -tocopherylquinone, alpha -tocopherylquinone an d ubiquinone-10, and inhibition was noncompetitive. Differential inhibition of neutral sphingomyelinase by antioxidants did not correlate with differe nt levels of protection against lipid peroxidation. The purified sphingomye linase was not inhibited significantly by ubiquinone-10 and ubiquinol-10, b ut ubiquinol-0 and ubiquinone-0 inhibited by 30 and 60% respectively. Our r esults demonstrate a direct inhibitory effect of ubiquinol on the plasma me mbrane n-SMase and support the participation of this molecule in the regula tion of ceramide-mediated signaling.