7] Regulatory subunits of bovine heart and rabbit skeletal muscle cAMP-dependent protein kinase isozymes

Abstract

Publisher Summary This chapter explains regulatory subunits of bovine heart and rabbit skeletal muscle cAMP-dependent protein kinase isozymes. Cyclic AMP-dependent protein kinase exists in two major isozymic forms, types I and II, which are present in varying relative concentrations, depending upon the tissue and species studied. Regulatory subunits from both isozymes can be isolated from a single tissue source when both types are present in approximately equal amounts, such as in porcine skeletal muscle. The choice of tissue and method of elution ultimately depends upon the quantity of R I or R II subunits needed and the nature of the studies for which they are to be used. The procedures described in the chapter for purification of R I and R II are based on relatively large amounts of starting material. If scaling down is appropriate, the final volume of cAMP-binding material which is eluted from diethylaminoethyl (DEAE)-cellulose may be small enough that concentration by (NH 4 ) 2 SO 4 before applying it to the affinity matrix is unnecessary. It is advisable to centrifuge all material to prevent blockage of the column. Omitting the (NH 4 ) 2 SO 4 precipitation step saves at least 6 hours and makes it possible to go from intact tissue to the cAMP-affinity column in