Hou W M, Zhang Z L, Tai H H
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Kentucky, Lexington.
Biochim Biophys Acta. 1988 Mar 4;959(1):67-75.
Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.
磷脂酰肌醇激酶从猪肝微粒体中溶解并纯化至表观均一。纯化步骤包括：用2% Triton X-100溶解微粒体、硫酸铵沉淀（20 - 35%饱和度）、活性蓝琼脂糖层析、DEAE - Sephacel层析以及连续两次羟基磷灰石层析。实现了4900倍的纯化，酶活性回收率为8%。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计该酶的分子量为55000。该酶受Mg2 +、Fe2 +、Mn2 +、Fe3 +和Co2 +刺激的程度依次降低。Ca2 +抑制Mg2 +刺激的活性，I50为0.4 mM。磷脂酰肌醇和ATP的表观Km值分别为120和60 microM。该酶受腺苷（I50 = 70 microM）、ADP（I50 = 120 microM）和槲皮素（I50 = 100 microM）抑制。该酶对巯基抑制剂也敏感。以纯化的酶作为免疫原，我们成功地在兔体内制备了针对磷脂酰肌醇激酶的抗体。在蛋白质印迹分析中，这些抗体似乎能识别来自各种猪组织的SDS - 聚丙烯酰胺凝胶电泳上Mr 55000的抗原。
