Existence of Mg2+-dependent, neutral sphingomyelinase in nuclei of rat ascites hepatoma cells.
A sphingomyelinase, which specifically hydrolyzes sphingomyelin into ceramide and phosphocholine, was solubilized from nuclear matrix fraction of rat ascites hepatoma, AH7974 cells. The solubilized enzyme was subjected to Mono Q column chromatography in an FPLC system. The sphingomyelinase which was adsorbed on the column and eluted at 0.25-0.5 M NaCl was characterized. The enzyme required 10 mM MgCl2, 0.01% Triton X-100, 1 mM dithiothreitol, and a higher concentration of buffer than 1 M for its maximal activity, and the optimal pH was 6.7-7.2 in 2 M Tris/acetic acid or 7.5 in 2 M potassium acetate/acetic acid. N-Ethylmaleimide completely inhibited the enzyme activity at 0.2 mM. Therefore, this enzyme is classified as a Mg2+-dependent, neutral sphingomyelinase. The sphingomyelinase sedimented at 4.3S through a 10-30% glycerol gradient containing 2 M potassium acetate. This enzyme was highly specific to sphingomyelin and did not hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. Various characteristics of the nuclear sphingomyelinase were similar to those of the plasma membrane enzyme except its requirement for a high concentration of buffer and SH-reagent.
Tamiya-Koizumi
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K
Laboratory of Cancer Cell Biology, Nagoya University School of Medicine, Aichi.
Umekawa
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H
Yoshida
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Kojima
K
K
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008274:Magnesium / Q000502:physiology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity
D010727:Phosphoric Diester Hydrolases; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium