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 cera mide 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 sphingo myelinase, we studied its relationship with enzymes present in mammalian ce lls and tissues, its subcellular localization, and properties that could be important for the regulation of its activity. Using specific antibodies, i t is suggested that the enzyme could represent one of several forms of neut ral sphingomyelinases present in the extract from brain particulate fractio n. 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 thi s enzyme. The molecular and enzymatic properties of the cloned neutral magn esium-dependent sphingomyelinase, produced using baculovirus or bacterial e xpression systems, have been analyzed, demonstrating the expected ion depen dence and substrate specificity. The enzyme activity also has a strong requ irement 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 properti es previously associated with neutral magnesium-dependent sphingomyelinases in crude or partially purified preparations.