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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., an...
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Published in: | The Journal of biological chemistry 1994-02, Vol.269 (6), p.4070-4077 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Subjects: | |
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Online Access: | Get full text |
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Summary: | 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/s0021-9258(17)41744-3 |