Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.
Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.
D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D008099:Liver / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D006656:Protamine Kinase / Q000378:metabolism; D051381:Rats; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D014599:Uterus / Q000378:metabolism
D000345:Affinity Labels; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D006656:Protamine Kinase