CHOLESTEROL 7-ALPHA-HYDROXYLASE ACTIVITIES FROM HUMAN AND RAT-LIVER ARE MODULATED IN-VITRO POSTTRANSLATIONALLY BY PHOSPHORYLATION DEPHOSPHORYLATION/

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 tripho sphate (ATP) in the presence and absence of bacterial alkaline phospha tase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinase. The amounts of P-32 incorporation after sep aration of human and rat C7 alpha H proteins by sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7 alph a H catalytic activities (determined by a radioisotope incorporation m ethod) and enzyme protein mass (determined by Western blotting and las er densitometry). Both human and rat C7 alpha H activities significant ly decreased after dephosphorylation by AP (-57%--72%) and increased u p to twofold with phosphorylation by rabbit muscle cAMP-dependent prot ein kinase. The increases in C7 alpha H activities were proportional t o the amounts of cAMP-dependent protein kinase used, and were coupled to P-32 incorporation into the purified enzymes, Both the activation o f 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-dep endent 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-inde pendent protein kinase. Rephosphorylation of the dephosphorylated C7 a lpha 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 pur ified enzymes. Bovine heart protein kinase was as potent as rabbit mus cle cAMP-dependent protein kinase in stimulating catalytic activity an d P-32 incorporation into the human C7 alpha H protein. Because the pr otein mass of these purified enzymes did not change, the short-term re gulation or catalytic efficiency of C7 alpha H (activity per protein m ass unit) is modulated, in vitro, posttranslationally by a phosphoryla tion/ dephosphorylation mechanism in both the human and the rat enzyme s.