Tryptic Peptide Mapping Studies on the Regulatory Subunits of Type II Protein Kinases from Cerebral Cortex and Heart. Evidence for Overall Structural Divergence and Differences in the Autophosphorylation and cAMP-binding Domains

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Abstract
Regulatory subunits of type II cAMP-dependent protein kinases (RII) (EC 2.7.1.37) from bovine brain and heart exhibit similar physicochemical and functional properties in vitro. The 2 forms of RII are markedly different in their antigenic determinants, cell and tissue distribution and subcellular localization. Each of these cAMP-binding proteins may possess some unique structural features. To assess the degree of overall divergence between the primary structures of brain RII and heart RII, tryptic peptides derived from the 2 proteins were mapped by reverse phase HPLC [high performance liquid chromatography] on a C18 column. When the column effluent was monitored at 280 nm, 15 peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. More complex HPLC profiles were observed by following peptide absorbance at 210 nm, but a similar level of diversity was apparent: 13 brain-RII-specific and 15 heart-RII-specific tryptic peptides were identified and resolved with a gradient (0-50%) of acetonitrile in 0.1% trifluoroacetic acid. In complementary experiments, classical 2-dimensional mapping analyses revealed that several 32P-labeled tryptic fragments derived from autophosphorylated and photoaffinity-labeled brain RII were separate and distinct from the 32P-peptides isolated from similarly treated heart RII. The HPLC mapping data document a structural basis for the immunological disparity between brain RII and heart RII and suggests the 2 cAMP-binding proteins are different proteins rather than interconvertible forms of a single protein. The 2-dimensional maps futher indicate that significant structural dissimilarities between brain RII and heart RRI also occur within the functionally conserved autophosphorylation and cAMP-binding domains.

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