Villalba M, Kelly K L, Mato J M
Fundacion Jimenez Diaz, Madrid, Spain.
Biochim Biophys Acta. 1988 Jan 18;968(1):69-76. doi: 10.1016/0167-4889(88)90045-6.
A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 microM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 microM myo-inositol, glucosamine, galactose, myo-inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylation of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.
一种糖脂已从大鼠肝细胞膜中纯化出来，并显示与从H35肝癌细胞中分离出的一种胰岛素敏感糖脂共同纯化。这种糖脂的极性头部基团是一种磷酸寡糖，由金黄色葡萄球菌的磷脂酰肌醇特异性磷脂酶C处理产生。有人提出，这种同样在胰岛素作用下产生的磷酸寡糖可能在胰岛素作用中发挥作用。用这种磷酸寡糖孵育环磷酸腺苷依赖性蛋白激酶的催化亚基，可抑制该激酶磷酸化组蛋白IIA、纯化的磷脂甲基转移酶制剂和肯普肽（一种磷酸接受肽）的活性。激酶活性的抑制呈剂量依赖性，当磷酸寡糖浓度约为2 microM时，组蛋白磷酸化受到50%的抑制。在高达1 mM的ATP浓度存在下证明了这种效应，表明磷酸寡糖在ATP的生理浓度下起作用，并且它不会与该核苷酸竞争激酶中的相同结合位点。磷酸寡糖对激酶活性的抑制可通过稀释或透析逆转，高达50 microM的肌醇、葡糖胺、半乳糖、肌醇1-磷酸、葡糖胺1-磷酸、半乳糖1-磷酸或磷酸胆碱均不能重现这种抑制作用。抑制活性对弱酸处理有抗性，但对碱处理、亚硝酸处理或高碘酸钠孵育不稳定。磷酸寡糖对大鼠脑蛋白激酶C对富含赖氨酸组蛋白的磷酸化以及环磷酸腺苷与环磷酸腺苷依赖性蛋白激酶的结合没有影响。总之，本研究中的数据表明，由胰岛素敏感糖脂产生的磷酸寡糖可能通过调节环磷酸腺苷依赖性蛋白激酶活性在胰岛素作用中发挥作用。
