Interconvertible cGMP-free and cGMP-bound forms of cGMP-dependent protein kinase in mammalian tissues.

Several vascular and nonvascular mammalian tissue extracts exhibited variable amounts of two peaks (peaks I and II) of cGMP-dependent protein kinase by NaCl elution of DEAE columns. When [3H]cGMP was added to the extracts before chromatography, a peak of protein-bound [3H]cGMP coeluted with peak II. [3H]cGMP was added to purified bovine lung cyclic nucleotide-free enzyme followed by chromatography on high performance liquid chromatography-DEAE. Two kinase peaks, the first of which represented mainly cGMP-free enzyme and the second of which represented cGMP-bound enzyme, eluted at the same positions as peaks I and II, respectively, of the crude extracts. The relative amount of peak II increased as a function of increasing the [3H]cGMP added before chromatography, and peak II could be converted partially to peak I by rechromatography. The holoenzyme is known to contain two slowly exchanging cGMP binding sites (sites 1) and two rapidly exchanging sites (sites 2). Some protein-bound [3H] cGMP found entirely in site 1 coeluted with peak I, although most of the enzyme in that peak was cGMP-free. When low [3H]cGMP was used for the initial incubation, relatively more of the protein-bound [3H] cGMP appeared in peak I and could represent binding of [3H]cGMP to only one of the two sites 1 of the kinase. The [3H]cGMP bound to the peak II enzyme completely filled both sites 1. Cyclic GMP binding to these sites caused the apparent conformational change which shifted the DEAE elution position of the enzyme. The peak II kinase was partially active and had a higher sensitivity to further cGMP activation of kinase than did the cGMP-free enzyme, suggesting that activation of kinase by binding of cGMP to site 2 was facilitated by prior binding at site 1. In fractions of the trailing edge of peak II, the kinase activity was virtually cGMP-independent, and both sites 1 and 2 were almost saturated with [3H]cGMP. These results suggested a further conformational change and direct increase in activity by binding of cGMP at site 2.

The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit.

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.