Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.
Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.
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凝溶胶蛋白对人血小板胞质磷酸肌醇-磷脂酶 C 同工酶的影响。
DOI：
10.1016/s0021-9258(19)50454-9
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Y. Nozawa
中科院分区：
文献类型：
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作者：
Y. Banno;T. Nakashima;T. Kumada;K. Ebisawa;Y. Nonomura;Y. Nozawa
用磷脂酰肌醇（PI）和磷脂酰肌醇4，5-二磷酸（PIP 2）对人血小板胞浆磷脂酰肌醇-磷脂酶C（PLC）进行了有效分离，结果表明，经Q-Sepharose和肝素-Sepharose柱层析，PLC有5个不同的活性峰。用抗PLC同工酶的各种抗体进行的Western印迹分析结果表明，在人血小板胞质溶胶中存在峰Ia（PLC-δ型）、峰Ib（PLC-γ 1型）和峰IIc（PLC-β型）以及两个未鉴定的活性峰（PLC-IIa和PLC-IIb）。将具有鸟苷5 ′-3-O-（硫代）三磷酸结合活性的蛋白质与PLC-IIa共洗脱，并纯化至均一。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定，其分子量分别为86和42 kDa，分别为凝溶胶蛋白（gelsolin）和肌动蛋白（actin）。在PLC-δ和PLC-γ 1级分中检测到大量的凝溶胶蛋白/肌动蛋白（1：1）复合物“凝溶胶蛋白复合物”。PLC-γ 1和凝溶胶蛋白复合物通过针对PLC-γ 1产生的抗体共免疫沉淀。此外，还发现部分纯化的牛脑PLC-γ 1级分与凝溶胶蛋白复合物缔合，并且通过加入1%胆酸钠来释放该缔合。这一发现促使我们研究凝溶胶蛋白复合物和游离凝溶胶蛋白对来自血小板胞质溶胶的上述PLC亚型的活性的影响。凝溶胶蛋白复合物不影响所有PLC异构体的PIP 2水解活性。相比之下，纯化的凝溶胶蛋白明显抑制PIP 2被PLC-Ia（δ）、PLC-Ib（γ 1）和PLC-IIa（未鉴定）水解，而对PLC-IIb（未鉴定）和PLC-IIc（β）的抑制作用是中等的。凝溶胶蛋白对PLC-γ 1水解PIP 2的抑制作用被大量的PIP 2底物减弱。这些结果表明凝溶胶蛋白对PLC的抑制是由于底物PIP 2通过其竞争性结合而被螯合。
The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex “gelsolin complex” were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.