Stubbs E B, Kelleher J A, Sun G Y
Department of Biochemistry, University of Missouri, Columbia 65203.
Biochim Biophys Acta. 1988 Feb 4;958(2):247-54. doi: 10.1016/0005-2760(88)90183-x.
Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [gamma-32P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150-200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4-11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40-70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP2 in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.
从大鼠大脑皮层分离并纯化的亚细胞组分，在存在内源性或外源性添加的脂质底物以及[γ-32P]ATP的情况下，检测磷脂酰肌醇（PI-）、磷脂酰肌醇-4-磷酸（PIP-）和二酰基甘油（DG-）激酶活性。在每个亚细胞组分中，包括胞质溶胶，均观察到了可测量的这三种激酶活性。然而，它们的亚细胞分布情况却独特各异。在没有外源性脂质底物的情况下，PI激酶的比活性在微粒体和非突触质膜组分中最高（150 - 200 pmol/分钟/毫克蛋白），而PIP激酶主要在突触体组分中具有活性（136 pmol/分钟/毫克蛋白）。基于总蛋白的百分比，回收的总PI激酶活性在胞质溶胶、突触体、微粒体和线粒体组分中最为丰富（4 - 11 nmol/分钟）。除微粒体组分外，当在外源性PIP存在的情况下进行检测时（在最终检测体积为0.1 ml时为4 nmol/20毫克蛋白），观察到PIP激酶活性具有类似的分布情况。外源性PIP（4 nmol/20毫克蛋白）在大多数组分中抑制PI激酶活性40 - 70%，同时增强PIP激酶活性。当分别在外源性添加PI或PIP的情况下进行检测时，在胞质溶胶组分中观察到了PI和PIP激酶活性，但在含有这些底物的热灭活膜中未观察到。当使用热灭活的富含DG的膜作为底物检测亚细胞组分的DG激酶活性时，DG激酶比活性主要存在于胞质溶胶中。然而，在脱氧胆酸盐存在的情况下孵育亚细胞组分，导致所有膜组分中的DG激酶活性显著增强。这些发现表明，所有三种脂质激酶在颗粒组分和可溶组分之间呈现双峰分布，每种激酶都表现出其自身独特的亚细胞拓扑结构。PIP激酶比活性在突触膜中的优先表达提示PIP₂参与突触功能，而PI激酶比活性在微粒体组分中的表达表明PIP在这些膜中具有其他尚未明确的功能。
