Characteristics and partial purification of a novel cytosolic, magnesium-independent, neutral sphingomyelinase activated in the early signal transduction of 1 alpha,25-dihydroxyvitamin D3-induced HL-60 cell differentiation.
Treatment of HL-60 cells with a 1 alpha,25-dihydroxyvitamin D3 induces activation of a neutral sphingomyelinase (SMase), resulting in a decrease in sphingomyelin (SM) levels and an increase in ceramide levels in a proposed "sphingomyelin cycle" of cell regulation (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). Cell-permeable synthetic ceramides induce HL-60 cell differentiation toward a monocytic lineage without conversion to sphingosine, suggesting that ceramide is a lipid mediator of cell differentiation (Okazaki, T., Bielawska, A., Bell, R., and Hannun, Y. (1990) J. Biol. Chem. 265, 15823-15831). In this study, we investigated a novel SMase that was activated 2-2.5 h after treatment of cells with 1 alpha,25-dihydroxyvitamin D3. The activated SMase was localized to the cytosolic fraction. It was inhibited by copper, ferric iron, and zinc and showed optimal activity at pH 7.5. A mixed micellar assay was developed for the enzyme, with optimal activity achieved at 12 mol% SM in Triton X-100 mixed micelles and at 20 mol% SM in deoxycholate micelles. The activity was modestly enhanced by phosphatidic acid, phosphatidylserine, or phosphatidylinositol, but not by other major phospholipids. Purification was performed by chromatography on DEAE anion-exchange, Q-Sepharose Fast Flow, hydroxylapatite, sphingosylphosphocholine affinity, and Superose 12 gel filtration columns. Two peaks of activity with molecular masses of 45 and 95 kDa were resolved by gel filtration chromatography on Superose 12. The specific activities of the purified 45- and 95-kDa enzymes were 2780 and 2790 nmol/mg/h, respectively. These data identify a novel cytosolic, magnesium-independent, neutral SMase(s) that is activated during cell differentiation.
Okazaki
T
T
Department of Medicine, Osaka Dental University, Japan.
Bielawska
A
A
Domae
N
N
Bell
R M
RM
Hannun
Y A
YA
D002117:Calcitriol / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D002853:Chromatography, Liquid; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008055:Lipids / Q000494:pharmacology; D008274:Magnesium / Q000502:physiology; D011480:Protease Inhibitors / Q000494:pharmacology; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D003902:Detergents; D008055:Lipids; D011480:Protease Inhibitors; D013439:Sulfhydryl Reagents; D013108:Sphingomyelin Phosphodiesterase; D002117:Calcitriol; D008274:Magnesium