Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes.
Concerted CMP-dependent [3H]inositol labeling of phosphoinositides and agonist activation of phospholipase C in rat brain cortical membranes.
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大鼠脑皮质膜中磷酸肌醇的协同 CMP 依赖性 [3H] 肌醇标记和磷脂酶 C 的激动剂激活。
DOI：
10.1111/j.1471-4159.1992.tb10958.x
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发表时间：
1992
影响因子：
4.7
通讯作者：
Fain,JN
中科院分区：
文献类型：
--
作者：
Claro,E;Wallace,MA;Fain,JN
在大鼠脑皮质膜中研究了磷酸肌醇的[3H]肌醇([3H]Ins)标记。 [3H]Ins 通过 CMP 依赖和独立机制被纳入共同的脂质池中。这些反应分别如下：（1）由磷脂酰肌醇（PtdIns）合酶催化的逆反应，以及（2）由PtdIns头基交换酶进行的反应。以 CMP 依赖性或独立方式预标记的膜磷酸肌醇可被鸟苷 5'-O-(3-硫代三磷酸) (GTPγS) 和卡巴胆碱刺激的磷脂酶 C 水解。然而，与 CMP 依赖性标记不同，1 mM (0.04%) 脱氧胆酸钠可抑制 CMP 独立的 [3H]Ins 掺入脂质。因此，当以一致的方式研究 PtdIns 标记和磷脂酶 C 刺激时，[3H]Ins 主要通过 Ptdlns 合酶催化反应掺入脂质中，因为观察磷脂酶 C 的卡巴胆碱刺激所需的脱氧胆酸盐的存在。几乎没有检测到 [3H]PtdIns 的直接分解，因为肌醇 1-单磷酸的产生很少，而且几乎没有检测到 [3H]PtdIns 的直接分解。肌-[3H]肌醇1,4-二磷酸酯是主要产品。尽管 PtdIns 标记和 3H 多磷酸肌醇形成不受 GTPγS 和卡巴胆碱的影响，并且没有或几乎没有滞后期，但 GTPγS 和卡巴胆碱刺激的 3H-Ins 磷酸盐的出现表现出明显的滞后（10 分钟）。此外，从 [3H]Ins 到 3H-Ins 磷酸盐的标记通量仅限于较窄的游离钙浓度范围 (10–300nM)。这些结果显示了 Ptdlns 合酶、Ptdlns 4-激酶和磷脂酶 C 的协同活性，并构成了一种简单的检测方法，用于使用 [3H]Ins 作为标记前体，对脑膜系统中的磷脂酶 C 进行鸟嘌呤核苷酸依赖性激动剂刺激。
[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP‐dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl‐inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP‐dependent or independent fashions were hydrolyzed by guan‐osine 5′‐O‐(3‐thiotriphosphate) (GTPγS)‐ and carbachol‐stimulated phospholipase C. Unlike CMP‐dependent labeling, however, CMP‐independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxy‐cholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the Ptdlns synthase‐catalyzed reaction because of the presence of deoxy‐cholate required to observe carbachol‐stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production ofmyo‐[3H]inositol 1‐mono‐ phosphate was minimal and myo‐[3H]inositol 1,4‐bisphos‐phate was the predominant product. Although PtdIns labeling and3Hpolyphosphoinositide formation were unaffected by GTPγS and carbachol and had no or little lag period, GTPγS‐ and carbachol‐stimulated appearance of3H‐Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to3H‐Ins phosphates was restricted to a narrow range of free calcium concentrations (10–300nM).These results show the concerted activities of Ptdlns synthase, Ptdlns 4‐kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide‐dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.
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