Acid Sphingomyelinase of Human Brain: Purification to Homogeneity

Abstract: Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human brain by extraction with 0.1% Triton X-100, followed by sequential chromatography on Concanavalin A-Sepharose, octyl-Sepharose, hydroxylapatite, DEAE-cellulose, red A-agarose, Sephadex G-200, and DEAE-cellulose with ampholyte elution. Sphingomyelinase activity was purified more than 20,000-fold from the starting homogenate with a 1% yield. Specific activity of up to 800 μmol/h/mg protein could be achieved. Gel electrophoresis with 6% polyacrylamide containing sodium dodecyl sulfate gave a single protein band with a molecular weight of 70,000, in good agreement with the molecular weight previously estimated from sucrose density gradient centrifugation in 0.1% Triton X-100. Triton X-100 could be readily removed from the enzyme by sucrose density gradient centrifugation. The Triton-free enzyme showed the same K _m and pH optimum. Heat stability of the enzyme was reversibly affected by Triton X-100, in that removal of the detergent made the enzyme more heat labile. The K _m of purified enzyme for sphingomyelin was 36 μ M . It was unaffected by sulfhydryl reagents, but was inhibited by dithiothreitol at high concentrations. The preparation was free of all lysosomal hydrolase activities tested, including galactosylceramidase and α-mannosidase, which tended to copurify in our previous procedure. The enzyme was inactive toward sphingosylphosphorylcholine. It was active with bis p -nitrophenyll- and bis4-methylumbelliferyl]phosphate and the chromogenic and fluorogenic sphingomyelin analogues.