Ghosh Mousumi, Smrcka Alan V
Department of Pharmacology and Physiology, University of Rochester School of Medicine, NY, USA.
Methods Mol Biol. 2004;237:67-75. doi: 10.1385/1-59259-430-1:67.
The activity of mammalian phosphoinositide-specific phospholipase C beta (PLC beta) is regulated by the alpha q family of G protein alpha subunits and by beta gamma subunits thought to be released from Gi. Interactions between G protein subunits and PLC beta can be assayed by measuring the stimulation of PLC beta enzymatic activity on reconstituting the purified G protein subunits with purified PLC beta on artificial phospholipid vesicles containing the substrate, phosphatidylinositol-4,5-bisphosphate (PIP2). These vesicles are doped with [3H]-inositol PIP2 and the rate of hydrolysis is determined by quantitating the amount of [3H]-inositol triphosphate (IP3) released from the vesicle into the aqueous phase. This assay provides a relatively simple method for assessing the activity PLC activity and its ability to be regulated by beta gamma and alpha(q) subunits. It can also be used to assess the functionality of the components after modification by mutagenesis, chemical modification, or in the presence of competing molecules.
哺乳动物磷酸肌醇特异性磷脂酶Cβ（PLCβ）的活性受G蛋白α亚基的αq家族以及被认为从Gi释放的βγ亚基的调节。G蛋白亚基与PLCβ之间的相互作用可以通过在含有底物磷脂酰肌醇-4,5-二磷酸（PIP2）的人工磷脂囊泡上用纯化的PLCβ重建纯化的G蛋白亚基时测量PLCβ酶活性的刺激来测定。这些囊泡用[3H] - 肌醇PIP2掺杂，水解速率通过定量从囊泡释放到水相中的[3H] - 肌醇三磷酸（IP3）的量来确定。该测定法提供了一种相对简单的方法来评估PLC活性及其受βγ和α（q）亚基调节的能力。它还可用于评估诱变、化学修饰后或存在竞争分子时组分的功能。
