At least two
isoenzymes of
sphingomyelinase (
sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal
acid sphingomyelinase and nonlysosomal
magnesium-dependent neutral
sphingomyelinase, catalyse the degradation of
sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of
sphingomyelin metabolism,
type A Niemann-Pick disease, is characterized by a deficiency of lysosomal
acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane
sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane
sphingomyelin in fibroblasts from a patient with
type A Niemann-Pick disease, which completely lack
acid sphingomyelinase activity but retain nonlysosomal neutral
sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane
sphingomyelin was labeled by incubating cells at low temperature with
phosphatidylcholine vesicles containing radioactive
sphingomyelin. A fluorescent analog of
sphingomyelin,
N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (
NBD-sphingomyelin) is seen to be readily transferred at low temperature from
phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]
sphingomyelin) to
NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]
sphingomyelin undergoes a similar transfer. The comparison of
sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated
sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]
sphingomyelin from the cell surface into the incubation medium. During long-term incubation, an increase in the formation of [14C]
ceramide correlating with the degradation of [14C]
sphingomyelin is observed in normal fibroblasts. In contrast, the level of [14C]
ceramide remains constant in Niemann-Pick diseased cells, which correlates with a higher level of intact [14C]
sphingomyelin remaining in these cells compared to normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)