A neutral magnesium-dependent sphingomyelinase isoform associated with intracellular membranes and reversibly inhibited by reactive oxygen species.
Activation of neutral sphingomyelinase(s) and subsequent generation of ceramide has been implicated in a wide variety of cellular responses. Although this enzyme(s) has not been purified and cloned from higher organisms, one mammalian cDNA has been previously isolated based on its similarity to the bacterial enzyme. To further elucidate the function of this neutral sphingomyelinase, we studied its relationship with enzymes present in mammalian cells and tissues, its subcellular localization, and properties that could be important for the regulation of its activity. Using specific antibodies, it is suggested that the enzyme could represent one of several forms of neutral sphingomyelinases present in the extract from brain particulate fraction. In PC12 cells, the enzyme is localized in the endoplasmic reticulum and is not present in the plasma membrane. The same result has been obtained in several cell lines transfected or microinjected with plasmids encoding this enzyme. The molecular and enzymatic properties of the cloned neutral magnesium-dependent sphingomyelinase, produced using baculovirus or bacterial expression systems, have been analyzed, demonstrating the expected ion dependence and substrate specificity. The enzyme activity also has a strong requirement for reducing agents and is reversibly inhibited by reactive oxygen species and oxidized glutathione. The studies demonstrate that the cellular localization and some properties of this enzyme are distinct from properties previously associated with neutral magnesium-dependent sphingomyelinases in crude or partially purified preparations.
Fensome
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AC
Cancer Research Campaign Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, United Kingdom.
Rodrigues-Lima
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F
Josephs
M
M
Paterson
H F
HF
Katan
M
M
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology; D015723:Gene Library; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism*; D008322:Mammals; D051379:Mice; D016716:PC12 Cells; D051381:Rats; D017382:Reactive Oxygen Species / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D007527:Isoenzymes; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium