Purification and characterization of neutral and acid sphingomyelinases from rat brain.
Neutral and acid sphingomyelinases were copurified from a rat brain P2 fraction by extraction with 1% Triton X-100, followed by (NH4)2SO4 fractionation, acetone powdering, extraction with 1% Triton X-100, (NH4)2SO4 fractionation, Sepharose CL-6B chromatography, and chromatofocusing. The neutral sphingomyelinase was eluted with buffer containing 0.4 M NaCl after the acid sphingomyelinase had been eluted with Polybuffer at pH 5.3. The neutral sphingomyelinase exhibited specific activity of 48,300 nmol/h/mg of protein, with 254-fold purification; the corresponding value for acid sphingomyelinase was 25,300 nmol/h/mg protein, with 668-fold purification from the P2 fraction. The purified neutral sphingomyelinase had no acid sphingomyelinase activity, and vice versa. The properties of the two enzymes were examined. A single band corresponding to a molecular weight of 67,000 was obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both enzymes. The pI was estimated to be 5.5 for both on isoelectric focusing. The native molecular weights of the neutral and acid sphingomyelinases were found to be 434,000 and 284,000, respectively, on gel filtration with Sepharose CL-6B. The single band obtained for each enzyme on SDS-PAGE was identified as an antigen with antibody raised against the purified neutral sphingomyelinase. Their amino acid compositions were very similar. The neutral and acid sphingomyelinases probably consist of common polypeptides and are immunologically cross-reactive.
Maruyama
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Division of Mental Retardation and Birth Defect Research, National Center of Neurology and Psychiatry, Tokyo, Japan.
Arima
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006358:Hot Temperature; D007526:Isoelectric Point; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium; D004229:Dithiothreitol