Goodwin C D, Cooper B W, Margolis S
J Biol Chem. 1982 Apr 25;257(8):4469-72.
Four lines of evidence presented here suggest that the activity of cholesterol 7 alpha-hydroxylase in rat liver is modulated by changes in its phosphorylation state. 1) Livers were homogenized and microsomes were isolated and washed in the presence of either 50 mM NaCl or 50 mM NaF, the latter an inhibitor of phosphoprotein phosphatases. The 7 alpha-hydroxylase activity of microsomes prepared with NaF was 80% greater than that of microsomes prepared with NaCl. 2) Incubation of 10,000 X g supernatants from rat liver for 20 min at 37 degrees C in the absence of 50 mM KF decreased the activity of microsomal cholesterol 7 alpha-hydroxylase by 52%. No significant change was seen in the presence of KF. 3) 7 alpha-Hydroxylase activity fell by 40% when microsomes were incubated with bacterial alkaline phosphatase compared to incubation of microsomes with phosphatase that was inhibited by phosphate and EDTA. 4) 7 alpha-Hydroxylase activity increased by 22% when phosphatase-treated microsomes were incubated for 40 min at 37 degrees C with 1 mM MgATP, 50 microM cAMP, and 200 units of cAMP-dependent protein kinase.
此处提供的四条证据表明，大鼠肝脏中胆固醇7α-羟化酶的活性受其磷酸化状态变化的调节。1）将肝脏匀浆，分离出微粒体，并在50 mM NaCl或50 mM NaF（后者是磷蛋白磷酸酶的抑制剂）存在的情况下进行洗涤。用NaF制备的微粒体的7α-羟化酶活性比用NaCl制备的微粒体高80%。2）在无50 mM KF的条件下，将大鼠肝脏10,000×g上清液在37℃孵育20分钟，微粒体胆固醇7α-羟化酶的活性降低了52%。在有KF的情况下未观察到显著变化。3）与用磷酸盐和EDTA抑制的磷酸酶孵育微粒体相比，将微粒体与细菌碱性磷酸酶孵育时，7α-羟化酶活性下降了40%。4）当用磷酸酶处理的微粒体在37℃与1 mM MgATP、50 microM cAMP和200单位的cAMP依赖性蛋白激酶孵育40分钟时，7α-羟化酶活性增加了22%。
