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 concentration-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 microg/ mL, 9.4 microg/mL, and 12.9 microg/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.
Kim
Seok Kyun
SK
Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, Dongjak-Ku, Seoul 156-756, Korea.
Jung
Sang Mi
SM
Ahn
Kyong Hoon
KH
Jeon
Hyung Jun
HJ
Lee
Dong Hun
DH
Jung
Kwang Mook
KM
Jung
Sung Yun
SY
Kim
Dae Kyong
DK
D028202:Abies; D031002:Acer; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects* / Q000201:enzymology; D002417:Cattle; D045744:Cell Line, Tumor; D002518:Ceramides / Q000096:biosynthesis; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020441:Ginkgo biloba; D006801:Humans; D007723:Korea; D008274:Magnesium; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D024301:Plant Bark; D010936:Plant Extracts / Q000494:pharmacology*; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism; D013997:Time Factors
D002518:Ceramides; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D010936:Plant Extracts; D013109:Sphingomyelins; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium