Nguyen L B, Shefer S, Salen G, Chiang J Y, Patel M
Department of Medicine and the Liver Center, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103, USA.
Hepatology. 1996 Dec;24(6):1468-74. doi: 10.1002/hep.510240628.
Purified cholesterol 7alpha-hydroxylases (C7alphaH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7alphaH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7alphaH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7alphaH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7alphaH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7alphaH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat liver C7alphaH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7alphaH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7alphaH proteins by cAMP-dependent protein kinase increased C7alphaH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7alphaH protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7alphaH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.
将来自人及大鼠肝脏微粒体以及转化大肠杆菌表达系统的纯化胆固醇7α-羟化酶（C7αH），在存在和不存在细菌碱性磷酸酶（AP）或兔肌肉腺苷3',5'-环磷酸（cAMP）依赖性蛋白激酶的情况下，与0.3 mmol/L [γ-32P]三磷酸腺苷（ATP）一起孵育。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离人及大鼠C7αH蛋白后，32P掺入量与C7αH催化活性（通过放射性同位素掺入法测定）和酶蛋白质量（通过蛋白质印迹法和激光密度测定法测定）相关。经AP去磷酸化后人及大鼠C7αH活性均显著降低（-57% - -72%），而经兔肌肉cAMP依赖性蛋白激酶磷酸化后活性增加至两倍。C7αH活性的增加与所用cAMP依赖性蛋白激酶的量成比例，并且与32P掺入纯化酶中相关。C7αH的激活和32P掺入量均呈时间依赖性，与5 U cAMP依赖性蛋白激酶孵育1小时后达到最大值。在第二组实验中，将纯化的人及大鼠肝脏C7αH与AP孵育30分钟进行去磷酸化，随后用抑制剂氟化钠使磷酸酶失活，再将C7αH与兔肌肉cAMP依赖性蛋白激酶或牛心cAMP非依赖性蛋白激酶孵育30分钟进行再磷酸化。经cAMP依赖性蛋白激酶对去磷酸化的C7αH蛋白进行再磷酸化后，C�αH催化活性增加至四倍，催化活性的刺激与32P掺入纯化酶中的增加平行。牛心蛋白激酶在刺激人C7αH蛋白的催化活性和32P掺入方面与兔肌肉cAMP依赖性蛋白激酶一样有效。由于这些纯化酶的蛋白质量没有变化，在体外，人及大鼠酶中C7αH（每单位蛋白质量的活性）的短期调节或催化效率通过磷酸化/去磷酸化机制进行翻译后调控。
