Comparison of adenosine 3':5'-monophosphate-dependent protein kinases from rabbit skeletal and bovine heart muscle.

F Hofmann,Ja Beavo,Pj Bechtel,Eg Krebs

doi:10.1016/s0021-9258(19)40885-5

Abstract

Homogeneous preparations of adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase from rabbit skeletal (Peak I) and bovine heart muscle have been compared. Each enzyme has an S20,w value of 7.0. Each enzyme binds 2 mol of cyclic AMP per mol of enzyme and is dissociated in the presence of saturating concentrations of cyclic AMP into a demeric regulatory subunit-cyclic AMP complex and two catalytic subunits. The isolated subunits recombine, resulting in the formation of the original holoenzyme in each case. Several differences between the two enzymes were found. Different salt concentrations are necessary for elution of the respective enzyme from DEAE-cellulose. Their regulatory subunits differ with respect to their sedimentation constants and mobility on sodium dodecyl sulfate gel electrophoresis. The regulatory subunit of the heart enzyme is rapidly phosphorylated by MgATP but this does not occur with the skeletal muscle enzyme. MgATP is bound with high affinity only to the skeletal muscle enzyme. The enzymes have different apparent dissociation constants and Hill coefficients for cyclic AMP binding. With the skeletal muscle enzyme MgATP increases the dissociation constants for cyclic AMP about 10-fold and decreases the Hill coefficient, while with the heart enzyme phosphorylation decreases the cissociation constant for cyclic AMP 5- to 6-fold and increases the Hill coefficient. Different concentrations of cyclic AMP are required to dissociate the skeletal and heart muscle enzymes. The presence of MgATP increases the concentration of cyclic AMP required to dissociate the skeletal muscle enzyme but decreases the concentration necessary to dissociate the heart enzyme.

Highlights

Homogeneous preparations of adenosine 3’:5’-monophosphate-dependent protein kinase from rabbit skeletal (Peak I) and bovine heart muscle have been compared
The most intensively studied enzymes have been the rabbit skeletal muscle protein kinase isolated from Peak I separated on DEAE-cellulose [6, 7, 15] and the bovine heart protein kinase [16,17,18,19,20]
Cyclic AMP-dependent protein kinase from bovine heart’ was prepared utilizing some steps taken from the method for the skeletal muscle enzyme [13] and one step taken from the procedure of Rubin et al [16]

Summary

Recipient of a postdoctoral fellowship from Muscular Dystrophy

Corbin et al [14] focused attention on the fact that these two types of enzyme can be distinguished by the ability of histones or high salt concentrations to alter their dependence on cyclic AMP. These effects are due to differences in the dissociability of the two kinds of protein kinase. The most intensively studied enzymes have been the rabbit skeletal muscle protein kinase isolated from Peak I separated on DEAE-cellulose [6, 7, 15] and the bovine heart protein kinase [16,17,18,19,20]. The possible benefits to be derived from such a study were increased by indications that these enzymes may serve as prototypes for two general isozymic forms of cyclic AMP-dependent protein kinase [21]

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DISCUSSION