Absence of high-affinity adenosine 3′,5′-monophosphate binding sites from the cytosol of three hepatic-derived cell lines

Abstract

Binding of [ 3H]adenosine 3′,5′-monophosphate (cAMP) to specific sites was compared in liver and three hepatic-derived, cultured cell lines, HTC, RLC, and H4-II-E. At physiological pH, there are small though significant differences in total binding, with liver >H4-II-E = RLC > HTC. No specific inhibitor of binding was demonstrated in the cultured cells. Scatchard analysis of binding suggested the presence of two sites in liver with k dl = 3.4 × 10 −8 m and K dll = ~ 10 −7 m. The cultured cell cytosols all lack the higher-affinity site but have a site similar to K d11 of liver, with K d = 3.5 × 10 −7 m. In all cell cytosols, basal protein kinase activity is higher than in liver, proportionally more of the kinase is cAMP independent, and the degree of cAMP stimulation of the kinase is much less than in liver. Ion exchange and exclusion chromatography revealed that the predominant protein kinase complex found in liver is virtually completely absent from HTC and RLC cells.