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

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

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

Hepatology

1996

1996-12

Purified cholesterol 7 alpha-hydroxylases (C7 alpha H) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-P-32] 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 P-32 incorporation after separation of human and rat C7 alpha H proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7 alpha H catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7 alpha H 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 C7 alpha H activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to P-32 incorporation into the purified enzymes, Both the activation of C7 alpha H and the amounts of P-32 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 C7 alpha H were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7 alpha H by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7 alpha H proteins by cAMP-dependent protein kinase increased C7 alpha H catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in P-32 incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and P-32 incorporation into the human C7 alpha H protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7 alpha H (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/ dephosphorylation mechanism in both the human and the rat enzymes.

bile-acid synthesis; cloning; dietary-cholesterol; enzyme; Escherichia coli; expression; Gastroenterology & Hepatology; hmg-coa reductase; messenger-rna levels; Phosphatase; purification

Journal Article

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1468-1474

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