Husebye E S, Flatmark T
Department of Biochemistry, University of Bergen, Norway.
Biochim Biophys Acta. 1989 Feb 9;1010(2):250-7. doi: 10.1016/0167-4889(89)90169-9.
A phosphatidylinositol-4-phosphate kinase (PIP kinase, EC 2.7.1.68) was purified about 1000-fold from the cytosolic fraction of bovine adrenal medulla by ammonium sulfate precipitation, anion exchange, phosphocellulose and gel permeation chromatography. The PIP kinase preparation was free from other polyphosphoinositide metabolizing activities, and some of its catalytic properties were studied using both membrane-bound and liposomal/micellar phosphatidylinositol 4-phosphate (PIP) as the substrate. Using a native substrate (chromaffin granule ghosts) enriched in PIP, the Km value for MgATP was about 130 microM at 5 mM Mg2+, and optimum activity was obtained at pH 7.2. ADP inhibited the PIP kinase activity in a mixed non-competitive manner. The enzyme was stimulated by Mg2+ and spermidine, whereas free calcium effectively inhibited the PIP kinase activity (I50 at about 0.1 microM Ca2+). This inhibition was independent of calmodulin and CaATP (80 microM) was not a substrate for the PIP kinase. The presence of near intracellular concentrations of potassium decreased the sensitivity to calcium ions (I50 of about 50 microM), but the inhibitory effect was still evident in the physiologically interesting concentration range. The concentration for half-maximal inhibition was three orders of magnitude higher (I50 of about 200 microM) using a liposomal/micellar suspension of purified PIP as the substrate, which demonstrates the importance of using a native substrate in studying the regulatory properties of this enzyme. It is concluded that the high sensitivity of the PIP kinase activity to calcium ions is likely to be physiologically significant, as recently proposed for the phosphatidylinositol (PI) kinase (Husebye, E.S. and Flatmark, T. (1988) Biochim. Biophys. Acta 968, 261-265), and may represent a negative feedback control of the cytosolic calcium concentration. The cationic amphiphile trifluoperazine was a potent inhibitor of PIP kinase activity (I50 at about 15 microM), and may represent a useful tool to study PI kinase selectively in membranes or cells containing both kinases.
通过硫酸铵沉淀、阴离子交换、磷酸纤维素和凝胶渗透色谱法，从牛肾上腺髓质的胞质部分中纯化出一种磷脂酰肌醇-4-磷酸激酶（PIP激酶，EC 2.7.1.68），纯化倍数约为1000倍。该PIP激酶制剂不具有其他多磷酸肌醇代谢活性，并以膜结合和脂质体/胶束形式的磷脂酰肌醇4-磷酸（PIP）作为底物研究了其一些催化特性。使用富含PIP的天然底物（嗜铬粒体质膜），在5 mM Mg2+条件下，MgATP的Km值约为130 μM，在pH 7.2时获得最佳活性。ADP以混合非竞争性方式抑制PIP激酶活性。该酶受到Mg2+和亚精胺的刺激，而游离钙有效抑制PIP激酶活性（在约0.1 μM Ca2+时I50）。这种抑制作用独立于钙调蛋白，并且80 μM的CaATP不是PIP激酶的底物。接近细胞内钾离子浓度的存在降低了对钙离子的敏感性（I50约为50 μM），但在生理相关浓度范围内抑制作用仍然明显。使用纯化PIP的脂质体/胶束悬浮液作为底物时，半数最大抑制浓度高三个数量级（I50约为200 μM），这表明在研究该酶的调节特性时使用天然底物的重要性。结论是，PIP激酶活性对钙离子的高敏感性可能具有生理意义，正如最近对磷脂酰肌醇（PI）激酶所提出的那样（Husebye, E.S.和Flatmark, T. (1988) Biochim. Biophys. Acta 968, 261 - 265），并且可能代表对胞质钙浓度的负反馈控制。阳离子两亲性药物三氟拉嗪是PIP激酶活性的有效抑制剂（I50约为15 μM），并且可能是在含有两种激酶的膜或细胞中选择性研究PI激酶的有用工具。
