Identification of three competitive inhibitors for membrane-associated, Mg2+-dependent and neutral 60 kDa sphingomyelinase activity

Methanol extracts of domestic plants of Korea were evaluated as a potential inhibitor of neutral pH optimum and membrane-associated 60 kDa sphingomyelinase (N-SMase) activity. In this study, we partially purified N-SMase from bovine brain membranes using ammonium sulfate. It was purified approximately 163-fold by the sequential use of DE52, Butyl-Toyopearl, DEAE-Cellulose, and Phenyl-5PW column chromatographies. The purified N-SMase activity was assayed in the presence of the plant extracts of three hundreds species. Based on the in vitro assay, three plant extracts significantly inhibited the N-SMase activity in a time- and concentrationn-dependent manner. To further examine the inhibitory pattern, a Dixon plot was constructed for each of the plant extracts. The extracts of Abies nephrolepis, Acer tegmentosum, and Ginkgo biloba revealed a competitive inhibition with the inhibition constant (Ki) of 11.9 mu g/mL, 9.4 mu g/mL, and 12.9 mu g/mL, respectively. These extracts also inhibited in a dose-dependent manner the production of ceramide induced by serum deprivation in human neuroblastoma cell line SH-SY5Y.