Characterization of acidic and neutral sphingomyelinase activities in crude extracts of HL-60 cells.
The enzymatic activities of acidic and neutral sphingomyelinases (aSMase and nSMase) in crude extracts of HL-60 cells prepared by short ultrasonic irradiation (sonicates) were characterized. It was found that although both have similar Km and Vmax (approximately 0.2 mM and approximately 3.5 nmol/mg per h, respectively), the two activities differ in many other aspects, including the following: (1) the aSMase activity has higher stability at 37 degrees C; (2) the aSMase is much less sensitive to Triton X-100 ( > 5 mM), compared with < or = 0.4 mM for the nSMase; (3) the nSMase, but not the aSMase, can discriminate between the natural bovine sphingomyelin substrate and the fluorescent substrate lissamine rhodamine dodecanoyl sphingosyl phosphocholine, suggesting that nSMase has higher substrate specificity. TNFalpha, which upon incubation with the HL-60 cells induces cellular SM hydrolysis, does not affect Km or Vmax of the nSMase in HL-60 sonicates. This suggests that TNFalpha may operate through translocation of either the enzyme or the substrate, thereby enhancing substrate availability and rate of hydrolysis, and not through enzyme activation. The relevance of these studies to the sphingomyelin cycle is discussed.
Samet
D
D
Laboratory of Membrane and Liposome Research, Department of Biochemistry, The Hebrew University, Hadassah School of Medicine, Jerusalem, Israel.
Barenholz
Y
Y
D000818:Animals; D002417:Cattle; D002452:Cell Count; D002457:Cell Extracts; D003902:Detergents / Q000737:chemistry; D004795:Enzyme Stability; D018922:HL-60 Cells / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D017830:Octoxynol / Q000737:chemistry; D012995:Solubility; D013010:Sonication; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013109:Sphingomyelins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002457:Cell Extracts; D003902:Detergents; D013109:Sphingomyelins; D014409:Tumor Necrosis Factor-alpha; D017830:Octoxynol; C112980:acid sphingomyelinase-1; C112981:acid sphingomyelinase-2; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium