CHARACTERIZATION AND COMPARISON OF MEMBRANE-ASSOCIATED AND CYTOSOLIC CAMP-DEPENDENT PROTEIN-KINASES - STUDIES ON HUMAN-ERYTHROCYTE PROTEIN-KINASES

Abstract

Cyclic[c]AMP-dependent protein kinase from human erythrocyte plasma membranes was solubilized with Triton X-100, partially purified and systematically characterized by a series of physicochemical studies. Sedimentation and gel filtration experiments showed that the 6.6 S holoenzyme had a Stokes radius (a) of 5.7 nm and was dissociated into native 4.8 S cAMP-binding (a = 4.5 nm) and 3.2 S catalytic (a = 2.6 nm) subunits. A minimum subunit MW of 48,000 was established for the regulatory subunit by photoaffinity labeling with 8-azido[32P]cAMP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. An asymmetric, tetrameric (R2C2) structure (MW .simeq. 160,000) was suggested for the membrane-derived enzyme. Membrane-derived protein kinase was characterized as a type I enzyme on the basis of its R subunit MW, pI [isoelectric point] values (R, 4.9; holoenzyme, 5.75 and 5.95), dissociation by 0.5 M NaCl and 50 .mu.g/ml of protamine, 20-fold reduced affinity for cAMP in the presence of 0.3 mM MgATP, elution from DEAE-cellulose at low ionic strength, and kinetic and cAMP-binding properties. The physicochemical properties of the membrane protein kinase closely parallel the characteristics of erythrocyte cytosolic protein kinase I but were clearly dissimilar from those of the soluble type II enzyme. Regulatory subunits of the membrane-associated and cytosolic type I kinases were indistinguishable in size, shape, subunit MW, charge, binding and reassociation properties, and peptide maps of the photoaffinity-labeled cAMP-binding site, suggesting a high degree of structural and functional homology in this pair of enzymes. In view of the predominant occurrence of particulate type II protein kinases in rabbit heart and bovine cerebral cortex, the distribution of membrane-associated protein kinases may be tissue- or species-specific, but not isoenzyme-specific.