Cholesterol 7α-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation

✍ Scribed by L B Nguyen; S Shefer; G Salen; J Y Chiang; M Patel

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 303 KB
• Volume: 24
• Category: Article
• ISSN: 0270-9139
• DOI: 10.1002/hep.510240628

✦ Synopsis

ciency of C7aH (activity per protein mass unit) is modu-Purified cholesterol 7a-hydroxylases (C7aH) from hulated, in vitro, posttranslationally by a phosphorylation/ man and rat liver microsomes, and from transformed dephosphorylation mechanism in both the human and Escherichia coli expression systems, were incubated the rat enzymes. (HEPATOLOGY 1996;24:1468-1474.) with 0.3 mmol/L [g-32 P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3,5-cyclic mono-

Cholesterol 7a-hydroxylase (C7aH) (EC 1.14.13.17) is the phosphate (cAMP)-dependent protein kinase. The first step and rate-limiting enzyme in the conversion of choamounts of 32 P incorporation after separation of human lesterol to bile acids in the liver. This enzyme has been and rat C7aH proteins by sodium dodecyl sulfate-polypurified by various laboratories, 3-6 its gene cloned, sequenced, acrylamide gel electrophoresis (SDS-PAGE) were relocalized to chromosome 8q11-q12, and partially characterlated to C7aH catalytic activities (determined by a radioized. [7][10] The catalytic activity of C7aH is regulated by a diurisotope incorporation method) and enzyme protein mass nal rhythm and various dietary, drug, and hormonal fac-(determined by Western blotting and laser densitometors. [12][14][15][16][17] The preferred substrate pool for C7aH is newly try). Both human and rat C7aH activities significantly synthesized cholesterol. 18 Therefore, bile acid synthesis is decreased after dephosphorylation by AP (057%-072%) regulated by the activity of both C7aH and 3-hydroxy-3and increased up to twofold with phosphorylation by methylglutaryl coenzyme A reductase, the rate-limiting enrabbit muscle cAMP-dependent protein kinase. The inzyme that controls the formation of endogenous cholescreases in C7aH activities were proportional to the terol. [15][16][17] The supply of cholesterol up-regulates C7aH activamounts of cAMP-dependent protein kinase used, and ity in the rat, 15,19 but inhibits it in the rabbit, 20 the hamster, 19 were coupled to 32 P incorporation into the purified enand the African Green monkey. 21 The reasons for such species zymes. Both the activation of C7aH and the amounts of differences have not been established. There is still a contro-32 P incorporation were time-dependent and reached a versy about mechanisms by which C7aH activity is controlled maximum after 1 hour of incubation with 5 U of cAMPby the enterohepatic flux of bile acids. 15,16 Earlier studies dependent protein kinase. In a second set of experihave suggested that C7aH activity could be regulated postments, purified human and rat liver C7aH were dephostranslationally by mechanisms involving cytosolic factors, 22 phorylated by 30-minute incubation with AP, followed disulfide bonds in the enzyme structure, 23 and phosphorylaby inactivation of the phosphatase by the inhibitor NaF, tion/dephosphorylation of the enzyme protein. [24][25] In contrast and rephosphorylation of C7aH by 30-minute incubation to 3-hydroxy-3-methylglutaryl coenzyme A reductase, which with rabbit muscle cAMP-dependent protein kinase or is deactivated by phosphorylation and stimulated by dephosbovine heart cAMP-independent protein kinase. Rephorylation, 29 it was earlier suggested that C7aH was deactiphosphorylation of the dephosphorylated C7aH provated by phosphatase-mediated dephosphorylation and stimteins by cAMP-dependent protein kinase increased ulated by a number of protein kinases. [24][25] However, C7aH catalytic activities up to fourfold, and the stimulaphosphorylation/dephosphorylation as a mechanism for posttion in catalytic activities paralleled the increases in 32 P translational regulation of C7aH activity is still disputed by incorporation into the purified enzymes. Bovine heart some investigators. protein kinase was as potent as rabbit muscle cAMP-

The objectives of this study are to (1) examine the effects dependent protein kinase in stimulating catalytic activof phosphorylation/dephosphorylation on catalytic activities ity and 32 P incorporation into the human C7aH protein. of various preparations of purified C7aH from human and Because the protein mass of these purified enzymes did rat liver; and (2) relate changes in catalytic activities under not change, the short-term regulation or catalytic effivarying phosphorylation/dephosphorylation conditions to the amounts of 32 P incorporated into the purified enzyme proteins.