Tooke N E, Hales C N, Hutton J C
Biochem J. 1984 Apr 15;219(2):471-80. doi: 10.1042/bj2190471.
Subcellular fractions were isolated from a rat beta-cell tumour by centrifugation of homogenates on Percoll and Urografin density gradients. Fractions were incubated with [gamma-32P]ATP, and labelling of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was used to measure phosphatidylinositol kinase and phosphatidylinositol 4-phosphate kinase activities, respectively. The distribution of enzyme markers in density gradients indicated that phosphatidylinositol kinase was located in both the plasma membrane and the secretory-granule membrane. Phosphatidylinositol 4-phosphate kinase activity was low in all fractions. Phosphatidylinositol kinase activity of secretory granules and plasma membranes was decreased to 10-20% of its initial value by raising the free [Ca2+] from 1 microM to 5 microM. The enzyme had a Km (apparent) for ATP of 110 microM (secretory granule) or 120 microM (plasma membrane) and a Ka for Mg2+ of 7 mM (secretory granule) or 6 mM (plasma membrane). Ca2+-sensitivity of phosphatidylinositol kinase in calmodulin-depleted secretory granules and plasma membranes was not affected by addition of exogenous calmodulin, although activity was stimulated by trifluoperazine in the presence of 0.1 microM or 40 microM-Ca2+. Trifluoperazine oxide had no effect on the enzyme activity of secretory granules. Plasma membranes had a phosphatidylinositol 4-phosphate phosphatase activity which was stimulated by raising the free [Ca2+] from 0.1 to 40 microM. The secretory granule showed no phosphatidylinositol 4-phosphate-degrading activity. These results suggest the presence in the tumour beta-cell of Ca2+-sensitive mechanisms responsible for the metabolism of polyphosphoinositides in the secretory granule and plasma membrane.
通过在Percoll和泛影葡胺密度梯度上对匀浆进行离心，从大鼠β细胞肿瘤中分离出亚细胞组分。将各组分与[γ-32P]ATP一起孵育，分别用磷脂酰肌醇4-磷酸和磷脂酰肌醇4,5-二磷酸的标记来测定磷脂酰肌醇激酶和磷脂酰肌醇4-磷酸激酶的活性。酶标志物在密度梯度中的分布表明，磷脂酰肌醇激酶位于质膜和分泌颗粒膜中。所有组分中磷脂酰肌醇4-磷酸激酶的活性都很低。通过将游离[Ca2+]从1微摩尔/升提高到5微摩尔/升，分泌颗粒和质膜的磷脂酰肌醇激酶活性降低到其初始值的10% - 20%。该酶对ATP的Km（表观）为110微摩尔/升（分泌颗粒）或120微摩尔/升（质膜），对Mg2+的Ka为7毫摩尔/升（分泌颗粒）或6毫摩尔/升（质膜）。在钙调蛋白缺失的分泌颗粒和质膜中，磷脂酰肌醇激酶的Ca2+敏感性不受外源钙调蛋白添加的影响，尽管在存在0.1微摩尔/升或40微摩尔/升Ca2+时，三氟拉嗪能刺激其活性。三氟拉嗪氧化物对分泌颗粒的酶活性没有影响。质膜具有一种磷脂酰肌醇4-磷酸磷酸酶活性，该活性通过将游离[Ca2+]从0.1微摩尔/升提高到40微摩尔/升而受到刺激。分泌颗粒未显示出磷脂酰肌醇4-磷酸降解活性。这些结果表明，肿瘤β细胞中存在对Ca2+敏感的机制，负责分泌颗粒和质膜中多磷酸肌醇的代谢。
