Determination of basal and stimulated levels of inositol triphosphate in [32P]orthophosphate-labeled platelets.
Determination of basal and stimulated levels of inositol triphosphate in [32P]orthophosphate-labeled platelets.
复制标题
测定[32P]正磷酸盐标记血小板中肌醇三磷酸的基础水平和刺激水平。
DOI：
10.1016/0003-2697(86)90007-2
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发表时间：
1986
影响因子：
2.9
通讯作者：
Smith,JB
中科院分区：
文献类型：
--
作者：
Dangelmaier,CA;Daniel,JL;Smith,JB
以前的研究表明，凝血酶刺激的血小板预标记的[3 H]肌醇或[32 P]正磷酸盐的结果增加放射性肌醇三磷酸，一种物质被认为是调节细胞内游离钙的水平。在本研究中，我们改进了从其他化合物，也与[32 P]正磷酸标记的酶处理和电泳相结合的决议肌醇三磷酸的方法。我们进一步证明了代谢ATP和磷脂酰肌醇二磷酸（肌醇三磷酸的前体）的比活性在[32 P]正磷酸标记的血小板中是相同的。因此，三磷酸肌醇的量与其在细胞代谢区室中的放射性成比例。用此方法测定了静息血小板和凝血酶刺激血小板中三磷酸肌醇的浓度分别为1-4和10-30 pmol/108个细胞。
Previous studies indicated that thrombin-stimulation of platelets prelabeled with [3H]inositol or [32P]orthophosphate results in an increase of radioactive inositol triphosphate, a substance thought to modulate the levels of free intracellular calcium. In the present study, we improved the method of resolution of inositol triphosphate from other compounds that are also labeled with [32P]orthophosphate using a combination of enzyme treatment and electrophoresis. We have further demonstrated that the specific activities of metabolic ATP and phosphatidylinositol diphosphate (the precursor of inositol triphosphate) are identical in [32P]orthophosphate-labeled platelets. It follows that the amount of inositol triphosphate is proportional to its radioactivity in the metabolic compartment of the cells. Using this protocol, the concentration of inositol triphosphate in resting and thrombin-stimulated platelets were determined to be 1–4 and 10–30 pmol/108cells, respectively.