Characterization of acidic and neutral sphingomyelinase activities in crude extracts of HL-60 cells

Dvorah Samet, Yechezkel Barenholz*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

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 K(m) and V(max) (~0.2 mM and ~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°C; (2) the aSMase is much less sensitive to Triton X-100 (>5 mM), compared with ≤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. TNFα, which upon incubation with the HL-60 cells induces cellular SM hydrolysis, does not affect K(m) or V(max) of the nSMase in HL-60 sonicates. This suggests that TNFα 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. Copyright (C) 1999 Elsevier Science Ireland Ltd.

Original languageEnglish
Pages (from-to)65-77
Number of pages13
JournalChemistry and Physics of Lipids
Volume102
Issue number1-2
DOIs
StatePublished - Nov 1999

Keywords

  • Acidic sphingomyelinase
  • HL-60 cells
  • Lissamine-rhodamine dodecanoyl sphingosyl phosphorylcholine
  • Neutral sphingomyelinase
  • Sphingomyelin
  • Sphingomyelin in cycle

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