The separate estimation of cAMP intracellularly bound to the regulatory subunits of protein kinase I and II in glucagon-stimulated rat hepatocytes.
A method is described for the separate determination of cAMP intracellularly bound to the regulatory moieties (RI and RII) of protein kinase I and II. The cAMP endogenously bound to RI or RII in hepatocyte extract was adsorbed to protein A-agarose beads coated with antibodies against RI or RII. The endogenously bound cAMP was eluted from the washed beads with dilute acetic acid before being assayed. By all criteria tested, the present method did not perturb the intracellularly established equilibrium between bound and free cAMP. Stabilization of R X cAMP complexes was achieved by including sulfate in the extraction medium and sulfate/glycerol during the subsequent steps. Hepatocytes were isolated from fed male rats and contained about 0.25 pmol of RI and 0.2 pmol of RII per 10(5) cells. An intracellular titration of the cAMP binding sites of RI and RII was achieved by incubating the cells with various concentrations (1 pM to 10 nM) of glucagon. The fractional saturation of RI and RII was always similar, being 20% in nonstimulated cells. 50% saturation occurred when free cAMP was 0.46 pmol/10(5) cells. A Scatchard plot of the data for the endogenous cAMP binding suggested that cAMP interacted with RI and RII in a slightly positively cooperative manner. About 5% of the intracellularly bound cAMP was sedimentable at 10,000 X g. The apparent affinity of these particulate-associated binding sites was similar to that of soluble RI and RII. Under the conditions used no evidence was obtained for cAMP binding to other proteins than RI and RII.
Ekanger
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R
Sand
T E
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Ogreid
D
D
Christoffersen
T
T
Døskeland
S O
SO
D000818:Animals; D001665:Binding Sites; D002451:Cell Compartmentation; D000242:Cyclic AMP / Q000378:metabolism*; D005934:Glucagon / Q000494:pharmacology*; D007424:Intracellular Fluid / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
D005934:Glucagon; D000242:Cyclic AMP; D011494:Protein Kinases