Adenosine-3':5'-monophosphate-binding proteins from bovine kidney. Isolation by affinity chromatography and limited proteolysis of the regulatory subunit of protein kinase II.
Affinity chromatography on cyclic AMP columns allowed a two-step isolation of the cyclic-AMP-binding proteins from bovine kidney cytosol. An AMP-binding protein (apparent molecular weight approximately 60 000) and large amounts of a low affinity binding protein ('P35'; apparent subunit size approximately 35 000) were obtained in practically pure form besides the high affinity binding proteins of the R type. Among the R proteins the dimer R2 of the regulatory subunit of protein kinase II (apparent subunit size approximately 54 000) represented the bulk material. Small amounts of monomer, of higher aggregates, and of a protein 'P49' (subunit size approximately 49 000) presumably identical with the regulatory subunit of protein kinase I were also detected. The R protein fraction of kidney also contained a high affinity binding protein of smaller size (designated as R'; molecular weight approximately 37 000) which appeared to be derived from protein R2 of protein kinase II by limited proteolysis. At all stages of purification, R protein and its aggregates could be quantitatively transformed into R' protein (or a closely related polypeptide) by several proteases including the relatively unspecific proteinase K. The degradation product exhibited unchanged cyclic-AMP-binding capacities but had largely lost the ability to inhibit the catalytic subunit C of protein kinase, to be phosphorylated by C, and to form a dimer. Preliminary experiments indicate that protein R' may be a natural component of kidney tissue.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D000242:Cyclic AMP / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D046911:Macromolecular Substances; D011494:Protein Kinases / Q000378:metabolism*
D002352:Carrier Proteins; D046911:Macromolecular Substances; D000242:Cyclic AMP; D011494:Protein Kinases