Rizzo M T, Weber G
Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis 46202-5200.
Cancer Res. 1994 May 15;54(10):2611-4.
The activity of 1-phosphatidylinositol 4-kinase (EC 2.7.1.67), the first committed ATP-utilizing enzyme of inositol 1,4,5-trisphosphate and diacylglycerol biosynthesis, was determined in a spectrum of rat hepatomas of different growth rates, in sarcoma, and in normal tissues of high cell renewal rates which include differentiating and regenerating liver. A standard isotopic method was developed to measure the enzymic activity in crude particulate extracts. In this assay, the enzyme activity was linear with time for 2 min and proportional with protein concentrations over a range of 0.1 to 1.0 mg per 0.1 ml reaction mixture. The optimum pH for both liver and hepatoma enzyme was 7.4. The apparent Km values of the kinase for ATP, Mg2+, and the substrate phosphatidylinositol in normal liver were 0.03, 10, and 0.2 mM, respectively, and in rapidly growing hepatoma 3924A 0.01, 0.1, and 5.3 mM. The kinase activity in adult rat livers was 0.3 to 0.5 +/- 0.01 nmol/h/mg protein. In hepatomas of slow and intermediate growth rates, kinase activity increased 5.3- to 7.6-fold, and in rapidly proliferating hepatoma 3924A, it was elevated 28.5-fold over that of normal liver. In rat sarcoma, kinase activity was 13.2-fold higher than in normal muscle. To clarify further the linkage between kinase activity and proliferation, enzymic activity was determined in rapidly growing rat tissues. The kinase activity in rat thymus, bone marrow, spleen, and testis increased 8.4-, 7.6-, 5.6- and 5.6-fold, respectively, over the values of normal rat liver; by contrast, in skeletal muscle, liver, heart, and renal cortex, the activities were low. In the rapidly growing neonatal rat liver and in 24-h regenerating liver, activities were 3.4- and 3.0-fold higher than in the adult resting liver. From this study, the relationship of 1-phosphatidylinositol 4-kinase activity with transformation and cell proliferation is clearly apparent in the markedly increased activity in transplantable hepatomas of different growth rates and in sarcoma and is further emphasized by the high activity observed in newborn and regenerating liver and in thymus, bone marrow, spleen, and testis. Since the kinase activity is linked with proliferation and malignancy, it may well be a sensitive target for chemotherapy.
1-磷脂酰肌醇4-激酶（EC 2.7.1.67）是肌醇1,4,5-三磷酸和二酰基甘油生物合成过程中首个消耗ATP的关键酶，我们测定了不同生长速率的一系列大鼠肝癌、肉瘤以及高细胞更新率的正常组织（包括分化和再生的肝脏）中该酶的活性。我们开发了一种标准同位素方法来测量粗制微粒提取物中的酶活性。在该测定中，酶活性在2分钟内与时间呈线性关系，并且在每0.1 ml反应混合物中蛋白质浓度为0.1至1.0 mg的范围内与蛋白质浓度成正比。肝脏和肝癌酶的最适pH均为7.4。正常肝脏中该激酶对ATP、Mg2+和底物磷脂酰肌醇的表观Km值分别为0.03、10和0.2 mM，而在快速生长的肝癌3924A中分别为0.01、0.1和5.3 mM。成年大鼠肝脏中的激酶活性为0.3至0.5 +/- 0.01 nmol/h/mg蛋白质。在生长缓慢和中等生长速率的肝癌中，激酶活性增加了5.3至7.6倍，而在快速增殖的肝癌3924A中，其活性比正常肝脏升高了28.5倍。在大鼠肉瘤中，激酶活性比正常肌肉高13.2倍。为了进一步阐明激酶活性与增殖之间的联系，我们测定了快速生长的大鼠组织中的酶活性。大鼠胸腺、骨髓、脾脏和睾丸中的激酶活性分别比正常大鼠肝脏的值增加了8.4、7.6、5.6和5.6倍；相比之下，骨骼肌、肝脏、心脏和肾皮质中的活性较低。在快速生长的新生大鼠肝脏和24小时再生肝脏中，活性分别比成年静止肝脏高3.4倍和3.0倍。从这项研究中可以明显看出，1-磷脂酰肌醇4-激酶活性与转化和细胞增殖之间的关系在不同生长速率的可移植肝癌和肉瘤中活性显著增加中清晰可见，并且在新生和再生肝脏以及胸腺、骨髓、脾脏和睾丸中观察到的高活性进一步强调了这一点。由于激酶活性与增殖和恶性肿瘤相关，它很可能是化疗的敏感靶点。
