Field F J, Henning B, Mathur S N
Biochim Biophys Acta. 1984 Nov 6;802(1):9-16. doi: 10.1016/0304-4165(84)90027-8.
The regulation of 3-hydroxy-3-methylglutarylcoenzyme A reductase and acylcoenzyme A:cholesterol acyltransferase activities by phosphorylation-dephosphorylation in rabbit intestine was studied in vitro. Preparing intestinal microsomes in the presence of 50 mM NaF caused a 64% decrease in the reductase activity. It had no effect on acyl-CoA:cholesterol acyltransferase activity. Microsomes that were prepared in NaF were incubated with intestinal cytosol, a partially purified phosphatase from cytosol, and Escherichia coli alkaline phosphatase. All three preparations increased 3-hydroxy-3-methylglutaryl-CoA reductase by two- or three-fold suggesting dephosphorylation and 'reactivation' of enzyme activity. Cytosol caused a 78% increase in acyl-CoA:cholesterol acyltransferase activity, but neither the partially purified phosphatase nor the E. coli alkaline phosphatase affected the acyltransferase activity. Microsomes incubated with increasing concentrations of MgCl2 and ATP decreased both the activities of 3-hydroxy-3-methylglutaryl-CoA reductase and acylcoenzyme A:cholesterol acyltransferase in a step-wise fashion. Whereas this inhibitory effect was specific for reductase, the effect on acyl-CoA:cholesterol acyltransferase activity was secondary to the presence of ATP in the assay mixture. The 8500 X g supernatant of intestinal whole homogenate from isolated intestinal cells or scraped mucosa was incubated with MgCl2, ATP and NaF. In microsomes prepared from this supernatant, the activity of 3-hydroxy-3-methylglutaryl-CoA reductase was significantly decreased. Again, no change was observed in the acyltransferase activity. The rate of cholesterol esterification in isolated intestinal cells was not affected by 0.1 mM cAMP or 50 mM NaF. We conclude that under conditions which regulate 3-hydroxy-3-methylglutaryl-CoA reductase activity in rabbit intestine by phosphorylation-dephosphorylation, no regulation of acyl-CoA:cholesterol acyltransferase activity is observed.
体外研究了兔肠道中3-羟基-3-甲基戊二酰辅酶A还原酶和酰基辅酶A：胆固醇酰基转移酶活性通过磷酸化-去磷酸化的调节作用。在50 mM氟化钠存在下制备肠道微粒体，导致还原酶活性降低64%。它对酰基辅酶A：胆固醇酰基转移酶活性没有影响。在氟化钠中制备的微粒体与肠道胞质溶胶、部分纯化的胞质溶胶磷酸酶和大肠杆菌碱性磷酸酶一起孵育。所有这三种制剂使3-羟基-3-甲基戊二酰辅酶A还原酶增加了两到三倍，表明酶活性去磷酸化并“重新激活”。胞质溶胶使酰基辅酶A：胆固醇酰基转移酶活性增加78%，但部分纯化的磷酸酶和大肠杆菌碱性磷酸酶均未影响酰基转移酶活性。用浓度不断增加的氯化镁和ATP孵育微粒体，以逐步方式降低3-羟基-3-甲基戊二酰辅酶A还原酶和酰基辅酶A：胆固醇酰基转移酶的活性。虽然这种抑制作用对还原酶具有特异性，但对酰基辅酶A：胆固醇酰基转移酶活性的影响是由于测定混合物中存在ATP所致。将来自分离的肠道细胞或刮下的黏膜的肠道全匀浆的8500×g上清液与氯化镁、ATP和氟化钠一起孵育。在此上清液制备的微粒体中，3-羟基-3-甲基戊二酰辅酶A还原酶的活性显著降低。同样，酰基转移酶活性未观察到变化。分离的肠道细胞中胆固醇酯化率不受0.1 mM环磷酸腺苷或50 mM氟化钠的影响。我们得出结论，在通过磷酸化-去磷酸化调节兔肠道中3-羟基-3-甲基戊二酰辅酶A还原酶活性的条件下，未观察到酰基辅酶A：胆固醇酰基转移酶活性的调节。
