Human platelets contain phospholipase C that hydrolyzes polyphosphoinositides.
Human platelets contain phospholipase C that hydrolyzes polyphosphoinositides.
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人血小板含有磷脂酶 C，可水解多磷酸肌醇。
DOI：
10.1073/pnas.80.17.5417
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发表时间：
1983
影响因子：
11.1
通讯作者：
S. Rittenhouse
中科院分区：
文献类型：
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作者：
S. Rittenhouse
已知受刺激的人血小板在磷脂酶c的激活下，会经历显著和快速的磷酸肌肽代谢变化。这种变化可能在血小板暴露于激动剂后促进Ca2+通量。我用被破坏的血小板检测了这种酶的活性。将人血小板超声处理，然后在Ca2+和脱氧胆酸存在的情况下，用4,5-二磷酸磷脂酰肌醇（PtdIns4,5P2）或4-单磷酸磷脂酰肌醇（PtdIns4P）孵育，这些底物发生明显的水解。通过阴离子交换和薄层色谱对水解产物的表征表明，这种活性的大部分是酶促的，可归因于磷脂酶c。在缺乏Ca2+或脱氧胆酸盐的情况下，只观察到磷酸单酯酶活性。我部分纯化了deae纤维素上的可溶性磷脂酶C，以尽量减少磷酸单酯酶的活性。在低盐浓度洗脱的馏分中，PtdIns4、5P2和PtdIns4P的磷脂酶C活性最高，磷酸单酯酶活性最低。这些组分中的酶在0.1 mM Ca2+和脱氧胆酸盐（1 mg/ml）存在时具有最大活性，并且显示出底物亲和力，其顺序为PtdIns大于PtdIns4P大于PtdIns4,5P2，最大速率为PtdIns4P大于PtdIns4,5P2大于PtdIns。这种底物偏好顺序似乎不同于在生理刺激的细胞中观察到的。讨论了造成这种差异的可能原因。
Stimulated human platelets are known to undergo marked and rapid changes in phosphoinositide metabolism consistent with the activation of phospholipase C. Such changes may promote a Ca2+ flux after platelets are exposed to agonists. I have examined this enzymatic activity by using disrupted platelets. When human platelets are sonicated and then incubated with phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) or phosphatidylinositol 4-monophosphate (PtdIns4P) in the presence of Ca2+ and deoxycholate, marked hydrolysis of these substrates occurs. Characterization of the hydrolysis products by anion exchange and thin-layer chromatography indicates that the bulk of this activity is enzymatic and attributable to phospholipase C. In the absence of Ca2+ or deoxycholate, only phosphomonoesterase activity is observed. I partially purified the soluble phospholipase C on DEAE-cellulose in order to minimize phosphomonoesterase activity. Fractions eluting at low salt concentrations contain the highest phospholipase C activity with respect to PtdIns4,5P2 and PtdIns4P and the lowest phosphomonoesterase activity. The enzyme(s) in these fractions is (are) maximally active in the presence of 0.1 mM Ca2+ and deoxycholate (1 mg/ml) and display(s) substrate affinities in the order PtdIns greater than PtdIns4P greater than PtdIns4,5P2 and maximum rates in the order PtdIns4P greater than PtdIns4,5P2 greater than PtdIns. This order of substrate preference appears to differ from that observed for physiologically stimulated cells. Possible reasons for such a discrepancy are discussed.