Identification, partial purification, and localization of a neutral sphingomyelinase in rabbit skeletal muscle: neutral sphingomyelinase in skeletal muscle.
We have investigated the presence of neutral sphingomyelinases present in rabbit skeletal muscle fractions. Neutral sphingomyelinase activity measurements and immunoblot analysis of various skeletal muscle fractions indicated that most of the neutral sphingomyelinase was associated with the junctional transverse tubules. Activity gel analysis of the detergent solubilized transverse tubule fraction revealed two distinct bands corresponding to molecular weight on the order of approximately 92 and 53 kDa. Moreover, monospecific antibody raised against pure neutral sphingomyelinase recognized both the 53 and the 92 kDa protein. Peptide mapping studies revealed that both neutral sphingomyelinase isoforms were similar. Moreover, both the enzymes catalyzed the hydrolysis of sphingomyelin to phosphocholine and ceramide. Lithium stimulated and Cu2+ inhibited the activity of both of the enzyme isoforms. However, the 53 kDa isoform was insensitive to activation by Mg2+, and thus differed from the 92 kDa isoform of neutral sphingomyelinase. The localization of neutral sphingomyelinase in skeletal muscle transverse tubule membrane is consistent with transverse tubule production of the sphingomyelin-derived second messenger, sphingosine. Since sphingosine has been shown to modulate calcium release from sarcoplasmic reticulum membranes (Sabbadini et al. (1992) J Biol Chem 207: 15473-15684), our work suggests that neutral sphingomyelinase/sphingosine signaling system may be a physiologically relevant regulator of calcium levels in skeletal muscle.
Ghosh
N
N
Department of Pediatrics, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA.
Sabbadini
R
R
Chatterjee
S
S
D000818:Animals; D015153:Blotting, Western; D002847:Chromatography, Agarose; D003300:Copper / Q000378:metabolism; D003902:Detergents / Q000378:metabolism; D008094:Lithium / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D018482:Muscle, Skeletal / Q000201:enzymology*; D010449:Peptide Mapping; D011817:Rabbits; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry* / Q000302:isolation & purification*; D014018:Tissue Distribution
D003902:Detergents; D003300:Copper; D008094:Lithium; D013108:Sphingomyelin Phosphodiesterase; D008274:Magnesium