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Papers In Press, published online ahead of print June 1, 2003
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Medicine, Rush Medical College, Chicago, IL 60612
Corresponding Author: psubbaia{at}rush.edu
Sphingomyelin (SM) and free cholesterol (FC) are concentrated in the plasma membranes of eukaryotes; however the physiological significance of their association is unclear. A common tool to study the role of cell membrane SM is digestion with bacterial sphingomyelinase (SMase) C, which hydrolyzes SM to ceramide and phosphorylcholine. However, it is not known whether the observed effects of SMase C treatment are due to the loss of SM per se, or due to the effects of ceramide, a powerful signaling molecule with multiple cellular effects. To address this, we tested SMase D from Corynebacterium, which hydrolyzes SM to ceramide phosphate, as an alternative probe. This enzyme specifically hydrolyzed SM in fibroblasts, without causing accumulation of ceramide. Treatment of fibroblasts with SMase D stimulated translocation of plasma membrane FC to intracellular sites by <20% of the rate observed with SMase C digestion. The increased esterification of FC in response to SMase C was not due to a direct effect of ceramide on FC esterification, because short chain ceramides inhibited the esterification both in intact cells and homogenates. The cells regenerated SM nearly completely within 5h after SMase C treatment. However, even after 20h, no regeneration was detected following SMase D digestion. These findings suggest that the majority of the translocation of plasma membrane FC caused by SMase C digestion is not due to the loss of SM. Rather, it is probably due to disruption of the membrane bilayer by ceramide, which is known to induce phase transitions. Ceramide phosphate does not appear to cause such disruption, and therefore SMase D may be a useful probe of membrane SM.
Revised on May 15, 2003
Accepted on May 20, 2003
Sphingomyelinase D, a novel probe for cellular sphingomyelin: Differential effects of sphingomyelinases C and D on cholesterol homeostasis in human skin fibroblasts
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