Adenosine 3',5'-monophosphate receptor proteins in HeLa cells.
The adenosine 3',5'-monophosphate receptor proteins of HeLa cells have been characterized. Using the Millipore filter assay, in the presence of 5'AMP and a phosphodiesterase inhibitor, specific [3H]cyclic AMP binding was detected in cytosol and in a nuclear-free particulate fraction, but not in nuclei. Both preparations exhibited biphasic Scatchard plots. 8-Azido[32P]cyclic AMP was used as a photoaffinity probe to covalently link ligand with receptor proteins. Proteins were then separated on denaturing gels and analyzed by autoradiography. The cytosol exhibited four specific binding proteins, with molecular weights of 46 000, 50 000, 52 000 and approx. 120 000. The 50 000/52 000 doublet could not be interconverted by phosphorylation-dephosphorylation reactions. On DEAE-cellulose, the 50 000-dalton protein eluted with peak II cyclic AMP-dependent protein kinase. The other proteins eluted with Peak I and with a binding peak not associated with kinase activity. Only the 50 000 protein was precipitated by type II protein kinase antibody from bovine heart. In the particulate fraction, the 120 000 protein was not detectable, but 8-azido[32P]cyclic AMP treatment revealed the other three proteins, with a relative increase in the 50 000-dalton protein. The results suggest that HeLa cells have four binding proteins which can associate with catalytic subunit and that the Peak I enzyme is heterogeneous, consisting of several distinct regulatory subunits.
D000249:Adenosine Monophosphate; D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010726:Phosphodiesterase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D046911:Macromolecular Substances; D010726:Phosphodiesterase Inhibitors; C017042:8-azidoadenosine-3',5'-monophosphate; D000249:Adenosine Monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases