Acid sphingomyelinase of human brain. Improved purification procedures and characterization.
Two new purification procedures for human brain sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12), one without any detergent and the other with Triton X-100, are described. These procedures were more readily reproducible than the earlier one from this laboratory (Yamaguchi, S., and Suzuki, K. (1977) J. Biol. Chem. 252, 3805-3813) and yield preparations of higher purity with the final specific activity in the range of 50 to 100 mumol/h/mg of protein. Although activities of most lysosomal hydrolases tested were effectively eliminated, substantial activities of galactosylceramidase and alpha-mannosidase remained in the purified preparation. They could be separated on the analytical scale from the sphingomyelinase activity by sucrose density gradient centrifugation. "Sphingomyelinase A" as we reported earlier appears to be aggregates of the sphingomyelinase (earlier designated as "sphingomyelinase B"). In Sephadex G-200 gel filtration, the enzyme showed an apparent molecular weight of 17 to 21 X 10(4). However, in the sucrose density gradient centrifugation in the presence of Triton X-100, it co-sedimented with bovine serum albumin (Mr = 67,000). Contrary to the earlier procedure, the new procedures eliminated the magnesium-dependent neutral sphingomyelinase activity from the acid sphingomyelinase preparations.
D001921:Brain / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D017830:Octoxynol; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification* / Q000378:metabolism
D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; D006867:Hydrolases; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase