The preparation of cyclic nucleotide-depleted regulatory subunits from type II adenosine 3':5'-monophosphate-dependent protein kinase by a nondenaturing method.
A nondenaturing method for the preparation of R subunits from type II cyclic AMP-dependent protein kinase is described. The procedure is based on the exchange of cyclic AMP, which is tightly bound to the R subunit, for more weakly bound cyclic GMP, which can be removed by washing and dialysis. Less than 5% of the available cyclic nucleotide-binding sites of R subunit prepared by this method contained cyclic AMP and less than 3% contained cyclic GMP. The C-subunit contamination (mol of C/mol of R monomer) was approximately 0.2%. These levels of contamination did not affect the properties of the R subunit as judged by (a) the ability of the R subunit to inhibit the activity of the C subunit and (b) the rate of exchange of cAMP into R2 . etheno-cAMP. The advantages of our method are that the protein is not subjected to denaturing conditions and that large amounts of material can be processed relatively rapidly.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D000242:Cyclic AMP / Q000302:isolation & purification; D006152:Cyclic GMP / Q000302:isolation & purification; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D009712:Nucleotides, Cyclic / Q000302:isolation & purification*; D011485:Protein Binding; D011489:Protein Denaturation; D011494:Protein Kinases / Q000032:analysis*
D009712:Nucleotides, Cyclic; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP