Possible regulation of phospholipase C activity in human platelets by phosphatidylinositol 4',5'-bisphosphate.
Possible regulation of phospholipase C activity in human platelets by phosphatidylinositol 4',5'-bisphosphate.
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磷脂酰肌醇 4,5-二磷酸可能调节人血小板中的磷脂酶 C 活性。
DOI：
10.1016/0003-9861(84)90071-7
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发表时间：
1984
影响因子：
3.9
通讯作者：
Schmid,HH
中科院分区：
文献类型：
--
作者：
Graff,G;Nahas,N;Nikolopoulou,M;Natarajan,V;Schmid,HH
研究发现，人血小板磷脂酶C在150 μ m ~ 5 μ m ca2 +浓度范围内可催化磷脂酰肌醇（PI）、磷脂酰肌醇4′-磷酸（DPI）和磷脂酰肌醇4′，5′-二磷酸（TPI）的依赖性降解，DPI和TPI均以浓度依赖性方式抑制[2-3 H]肌醇标记PI （250 μ m）的水解。牛肉脑中的DPI和TPI的脂肪酸组成与大豆PI不同，使用它们可以评估多磷酸肌苷对磷脂酶c水解PI的抑制作用。脂肪酸分析所形成的二酰基甘油表明，DPI和TPI与PI混合孵育时，是PI水解的竞争底物。将DPI - PI比值从0提高到0.3，使PI向DPI的降解发生转变，但对1,2 -二酰基甘油的形成没有太大影响。单独的TPI，或与PI混合，是磷脂酶c的差底物。另一方面，将TPI PI比率从0增加到0.21，抑制PI降解（大于等于95%）和1,2 -二酰基甘油（大于等于82%）的总体形成。动力学分析表明，TPI是一种混合型抑制剂，其Ki约为10 μ m，当TPI （36 μ m）与PI （250 μ m）混合时，ca2 +在PI水解过程中的K a从5 μ m显著提高到180 μ m。在这些条件下，只有当钙浓度接近4 m m时，才能达到最佳的PI降解。对来自五个不同供体的未刺激人血小板的磷脂的分析显示，DPI PI和TPI PI比值分别为0.42和0.16。这些发现，结合观察到的TPI在TPI PI比率为0.16时对PI水解的抑制，将表明在未刺激的血小板中磷脂酶C活性可能被大于或等于75%的人抑制。在凝血酶刺激下，完整血小板中33 P预标记磷脂的变化表明TPI和DPI的33 P标记均短暂下降（15 s），随后[33 P]磷脂酸升高，但[33 P] PI未发生变化。发现DPI在与PI的混合物中被磷脂酶C选择性地降解，其DPI PI比例确定存在于未受刺激的血小板中，这表明DPI在1,2 -二酰基甘油的形成中可能比PI更重要，后者被认为是用于血栓素生物合成的花生四烯酸的前体。此外，结果表明，在人血小板中，TPI可能是肌醇磷脂形成1,2 -二酰基甘油的调节剂。
Phospholipase C from human platelets was found to catalyze the Ca 2+-dependent degradation of phosphatidylinositol (PI), phosphatidylinositol 4′-phosphate (DPI), and phosphatidylinositol 4′, 5′-bisphosphate (TPI) at Ca 2+ concentrations from 150 μ m to 5 m m. Both DPI and TPI inhibited the hydrolysis of [2-3 H] inositol-labeled PI (250 μ m) in a concentration-dependent manner. The use of DPI and TPI from beef brain, both of which have fatty acid compositions different from that of soybean PI, permitted an assessment of the inhibitory effect of polyphosphoinositides on the hydrolysis of PI by phospholipase C. Fatty acid analysis of the diacylglycerols formed demonstrated that DPI and TPI, when incubated in mixture with PI, were competitive substrates for PI hydrolysis. Increasing the DPI PI ratio from 0 to 0.3 caused a shift in the degradation of PI to DPI without greatly affecting the formation of 1, 2-diacylglycerol. TPI alone, or in mixture with PI, was a poor substrate for phospholipase C. Increasing the TPI PI ratio from 0 to 0.21, on the other hand, inhibited both PI degradation (⩾ 95%) and overall formation of 1, 2-diacylglycerol (⩾ 82%). Kinetic analysis revealed that TPI acts as a mixed-type inhibitor with a K i of about 10 μ m. The K a for Ca 2+ in PI hydrolysis was profoundly increased from 5 to 180 μ m when TPI (36 μ m) was included with PI (250 μ m). Optimum PI degradation under these conditions was only attained when the calcium concentration approached 4 m m. Analysis of phospholipids from unstimulated human platelets from five different donors revealed DPI PI and TPI PI ratios of 0.42 and 0.16, respectively. These findings, combined with the observed inhibition of PI hydrolysis by TPI at a TPI PI ratio of 0.16, would suggest that in unstimulated platelets phospholipase C activity may be inhibited by⩾ 75%. Changes in 33 P-prelabeled phospholipids of intact platelets upon stimulation with thrombin indicated a transient decline in 33 P label of both TPI and DPI (15 s) followed by an increase in [33 P] phosphatidic acid but no change in [33 P] PI. The finding that DPI is selectively degraded by phospholipase C in mixture with PI at DPI PI ratios determined to be present in unstimulated platelets indicates that DPI may be more important than PI in the formation of 1, 2-diacylglycerol which is believed to serve as precursor of arachidonic acid for thromboxane biosynthesis. Furthermore, the results suggest that in human platelets TPI may serve as modulator for the formation of 1, 2-diacylglycerol from inositol phospholipids.