Studies on the Adenosine 3′, 5′-Monophosphate-dependent Protein Kinases of Rabbit Skeletal Muscle

Experiments were carried out which showed that the R subunits can be readily altered in vitro by aging or by proteolytic attack, so it is possible that some of the forms that were detected may have been formed during enzyme isolation.

Abstract

Abstract The adenosine 3',5'-monophosphate-dependent (cAMP-dependent) protein kinases of rabbit skeletal muscle have been partially characterized in terms of molecular size and subunit composition. Three and possibly four species of protein kinase have been detected. The largest form of the enzyme was estimated by gel filtration and sedimentation to have a molecular weight of 123,000 and to be made up of a catalytic subunit (C) and a regulatory or cAMP-binding subunit (R) with molecular weights of 49,000 and 82,000, respectively. A second muscle protein kinase was estimated to have a molecular weight of 76,000 with a catalytic subunit the same size as that of the larger protein kinase and with an R subunit having a molecular weight of 50,000. The other muscle protein kinases were not purified sufficiently to permit making meaningful estimates of their molecular weights, but it could be concluded that they differed from one another primarily in the size of their R subunits. Experiments were carried out which showed that the R subunits can be readily altered in vitro by aging or by proteolytic attack, so it is possible that some of the forms that were detected may have been formed during enzyme isolation. Resolution of the muscle protein kinases into C subunit and R·[3H]cAMP complex fractions was achieved by substrate-affinity chromatography after dissociation of the holoenzymes in the presence of [3H]-cAMP. Reconstitution of the original enzyme forms from separated C and R subunits was shown by physical techniques and kinetic measurements.