Kelly K L, Merida I, Wong E H, DiCenzo D, Mato J M
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Biol Chem. 1987 Nov 5;262(31):15285-90.
Addition of isoproterenol to isolated rat adipocytes prelabeled with [32P]phosphate caused an increase in the phosphorylation and activation of phospholipid methyltransferase. 32P-Labeled phospholipid methyltransferase was recovered by immunoprecipitation and gel electrophoresis. Analysis of 32P-labeled peptides revealed one site of phosphorylation regulated by isoproterenol, and analysis of phosphoamino acids demonstrated that the incorporation of [32P]phosphate was on phosphoserine. Incubation of adipocytes with isoproterenol in the presence of insulin or a phospho-oligosaccharide inhibited the phosphorylation and activation of this enzyme. The inhibitory effect of insulin on the phosphorylation of phospholipid methyltransferase was reversible, and it was mimicked by a phospho-oligosaccharide. The phospho-oligosaccharide was generated by hydrolysis of an isolated glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The insulin-like effect of this phospho-oligosaccharide on the phosphorylation of phospholipid methyltransferase was demonstrated in isolated adipocytes, and the effect was abolished by treatment of the phospho-oligosaccharide with 10% NH4OH, nitrous acid, or sodium periodate. These data suggest that in intact adipocytes the effect of insulin to inhibit the phosphorylation/activation of phospholipid methyltransferase is mediated by a phospho-oligosaccharide generated by a phosphatidylinositol-specific phospholipase C.
向预先用[32P]磷酸盐标记的离体大鼠脂肪细胞中添加异丙肾上腺素，会导致磷脂甲基转移酶的磷酸化和活性增加。通过免疫沉淀和凝胶电泳回收32P标记的磷脂甲基转移酶。对32P标记的肽段进行分析，揭示了一个受异丙肾上腺素调节的磷酸化位点，对磷酸氨基酸的分析表明，[32P]磷酸盐掺入到了磷酸丝氨酸上。在胰岛素或磷酸寡糖存在的情况下，用异丙肾上腺素孵育脂肪细胞可抑制该酶的磷酸化和活性。胰岛素对磷脂甲基转移酶磷酸化的抑制作用是可逆的，并且可被磷酸寡糖模拟。该磷酸寡糖是通过用金黄色葡萄球菌的磷脂酰肌醇特异性磷脂酶C水解分离的糖脂产生的。这种磷酸寡糖对磷脂甲基转移酶磷酸化的胰岛素样作用在离体脂肪细胞中得到证实，并且用10%氢氧化铵、亚硝酸或高碘酸钠处理该磷酸寡糖后，该作用消失。这些数据表明，在完整的脂肪细胞中，胰岛素抑制磷脂甲基转移酶磷酸化/活性的作用是由磷脂酰肌醇特异性磷脂酶C产生的磷酸寡糖介导的。
