Varsányi M, Messer M, Brandt N R, Heilmeyer L M
Biochem Biophys Res Commun. 1986 Aug 14;138(3):1395-404. doi: 10.1016/s0006-291x(86)80438-7.
Incubation of rabbit skeletal muscle microsomes or isolated triads with gamma 32P-ATP/Mg2+ in the absence and in the presence of added phosphatidylinositol resulted in the formation of phosphatidylinositol 4-phosphate catalyzed by phosphatidylinositol kinase. When phosphatidylinositol 4-phosphate was added as exogenous substrate, phosphatidylinositol 4,5-bisphosphate was also formed demonstrating the presence of a membrane bound phosphatidylinositol 4-phosphate kinase. Triads were broken mechanically in a French press and separated on a continuous sucrose gradient. Incubation of these fractions with gamma 32P-ATP/Mg2+ resulted in a rapid labeling of phospholipid in a membrane fraction banding between transverse tubules and the terminal cisternae. Partial triad breakage and triad reformation experiments indicated that this phosphatidylinositol kinase was associated with T-tubules. When exogenous phosphatidylinositol 4-phosphate was employed as substrate phosphatidylinositol 4,5-bisphosphate and phosphatidic acid were formed, indicating the presence of all the enzymes of the polyphosphoinositide signaling system in this special membrane fraction. In contrast, heart muscle microsomes or plasma membranes can catalyze this reaction sequence from endogenous formed phosphatidylinositol 4-phosphate.
在不存在和存在添加的磷脂酰肌醇的情况下，将兔骨骼肌微粒体或分离的三联体与γ-32P-ATP/Mg2+一起温育，导致磷脂酰肌醇激酶催化形成磷脂酰肌醇4-磷酸。当添加磷脂酰肌醇4-磷酸作为外源底物时，也形成了磷脂酰肌醇4,5-二磷酸，这表明存在膜结合的磷脂酰肌醇4-磷酸激酶。三联体在法国压榨机中机械破碎，并在连续蔗糖梯度上分离。将这些组分与γ-32P-ATP/Mg2+一起温育，导致在横小管和终末池之间的膜组分条带中磷脂快速标记。部分三联体断裂和三联体重组实验表明，这种磷脂酰肌醇激酶与T小管相关。当使用外源磷脂酰肌醇4-磷酸作为底物时，形成了磷脂酰肌醇4,5-二磷酸和磷脂酸，表明在这个特殊的膜组分中存在多磷酸肌醇信号系统的所有酶。相比之下，心肌微粒体或质膜可以从内源性形成的磷脂酰肌醇4-磷酸催化这个反应序列。
