Imai A, Rebecchi M J, Gershengorn M C
Biochem J. 1986 Dec 1;240(2):341-8. doi: 10.1042/bj2400341.
Regulation of phosphatidylinositol kinase (EC 2.7.1.67) and phosphatidylinositol 4-phosphate (PtdIns4P) kinase (EC 2.7.1.68) was investigated in highly enriched plasma-membrane and cytosolic fractions derived from cloned rat pituitary (GH3) cells. In plasma membranes, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] added exogenously enhanced incorporation of [32P]phosphate from [gamma-32P]MgATP2- into PtdIns(4,5)P2 and PtdIns4P to 150% of control; half-maximal effect occurred with 0.03 mM exogenous PtdIns(4,5)P2. Exogenous PtdIns4P and phosphatidylinositol (PtdIns) had no effect. When plasma membranes prepared from cells prelabelled to isotopic steady state with [3H]inositol were used, there was a MgATP2- dependent increase in the content of [3H]PtdIns(4,5)P2 and [3H]PtdIns4P that was enhanced specifically by exogenous PtdIns(4,5)P2 also. Degradation of 32P- and 3H-labelled PtdIns(4,5)P2 and PtdIns4P within the plasma-membrane fraction was not affected by exogenous PtdIns(4,5)P2. Phosphoinositide kinase activities in the cytosolic fraction were assayed by using exogenous substrates. Phosphoinositide kinase activities in cytosol were inhibited by exogenously added PtdIns(4,5)P2. These findings demonstrate that exogenously added PtdIns(4,5)P2 enhances phosphoinositide kinase activities (and formation of polyphosphoinositides) in plasma membranes, but decreases these kinase activities in cytosol derived from GH3 cells. These data suggest that flux of PtdIns to PtdIns4P to PtdIns(4,5)P2 in the plasma membrane cannot be increased simply by release of membrane-associated phosphoinositide kinases from product inhibition as PtdIns(4,5)P2 is hydrolysed.
在源自克隆大鼠垂体（GH3）细胞的高度富集的质膜和胞质组分中，研究了磷脂酰肌醇激酶（EC 2.7.1.67）和磷脂酰肌醇4-磷酸（PtdIns4P）激酶（EC 2.7.1.68）的调节。在质膜中，外源添加的磷脂酰肌醇4,5-二磷酸[PtdIns(4,5)P2]可将[γ-32P]MgATP2-中的[32P]磷酸掺入PtdIns(4,5)P2和PtdIns4P的量提高至对照的150%；0.03 mM外源PtdIns(4,5)P2时出现半数最大效应。外源PtdIns4P和磷脂酰肌醇（PtdIns）无作用。当使用用[3H]肌醇预标记至同位素稳态的细胞制备的质膜时，[3H]PtdIns(4,5)P2和[3H]PtdIns4P的含量有MgATP2-依赖性增加，外源PtdIns(4,5)P2也可特异性增强此增加。质膜组分中32P和3H标记的PtdIns(4,5)P2和PtdIns4P的降解不受外源PtdIns(4,5)P2影响。通过使用外源底物测定胞质组分中的磷酸肌醇激酶活性。外源添加的PtdIns(4,5)P2可抑制胞质溶胶中的磷酸肌醇激酶活性。这些发现表明，外源添加的PtdIns(4,5)P2可增强质膜中的磷酸肌醇激酶活性（以及多磷酸肌醇的形成），但可降低源自GH3细胞的胞质溶胶中的这些激酶活性。这些数据表明，质膜中从PtdIns到PtdIns4P再到PtdIns(4,5)P2的通量不能简单地通过PtdIns(4,5)P2水解时膜相关磷酸肌醇激酶从产物抑制中释放来增加。
