Ghosh N, Sabbadini R, Chatterjee S
Department of Pediatrics, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA.
Mol Cell Biochem. 1998 Dec;189(1-2):161-8. doi: 10.1023/a:1006910200656.
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.
我们研究了兔骨骼肌组分中中性鞘磷脂酶的存在情况。对各种骨骼肌组分进行中性鞘磷脂酶活性测定和免疫印迹分析表明，大部分中性鞘磷脂酶与连接性横管相关。对经去污剂溶解的横管组分进行活性凝胶分析，发现两条不同的条带，其分子量约为92 kDa和53 kDa。此外，针对纯中性鞘磷脂酶产生的单特异性抗体识别53 kDa和92 kDa的蛋白质。肽图谱研究表明，两种中性鞘磷脂酶同工型相似。而且，这两种酶都催化鞘磷脂水解为磷酸胆碱和神经酰胺。锂刺激而Cu2+抑制这两种酶同工型的活性。然而，53 kDa同工型对Mg2+激活不敏感，因此不同于92 kDa的中性鞘磷脂酶同工型。中性鞘磷脂酶在骨骼肌横管膜中的定位与鞘磷脂衍生的第二信使鞘氨醇在横管中的产生一致。由于鞘氨醇已被证明可调节肌浆网膜钙释放（Sabbadini等人，(1992) J Biol Chem ２０７：15473 - 15684），我们的工作表明中性鞘磷脂酶/鞘氨醇信号系统可能是骨骼肌中钙水平的生理相关调节因子。
