Partial purification and properties of acid sphingomyelinase from rat liver.
Acid sphingomyelinase was purified approximately 5,200-fold from the mitochondria-lysosome-enriched particles of rat liver by sequential chromatography on DEAE-cellulose, octyl-Sepharose, Sephacryl S-300, Concanavalin A-Sepharose, and CM-cellulose. The specific activity of this highly purified enzyme was 3.2 mmol per hr per mg protein. The enzyme was active against 2-hexadecanoylamino-4-nitrophenylphosphorylcholine, but bis-4-methylumbelliferyl-phosphate and bis-p-nitrophenyl-phosphate were poor substrates. The preparation was free of Mg2+-dependent neutral sphingomyelinase and eight lysosomal enzymes except for the trace amount of acid phosphatase and beta-galactosidase. Apparent molecular weight of the enzyme was 200,000, estimated by Sephadex G-200 filtration in 0.1% Triton X-100. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed three major bands corresponding to molecular weights of 45,600, 44,500, and 40,000 with several minor bands. Characterization of the enzyme revealed almost the same properties as those of human tissues reported by other investigators, including pH optimum, requirement of Triton X-100, effects of metal divalent cations, phosphate ion, EDTA, some thiol blocking reagents, and amphophilic drugs.
D000818:Animals; D005260:Female; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D001371:trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride / Q000494:pharmacology
D001371:trans-1,4-Bis(2-chlorobenzaminomethyl)cyclohexane Dihydrochloride; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase