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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
Author(s) -
Stein Jill C.,
Sarkar Dwijen,
Rubin Charles S.
Publication year - 1984
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1984.tb02712.x
Subject(s) - peptide , biochemistry , autophosphorylation , protein kinase a , chromatofocusing , chemistry , biology , kinase , size exclusion chromatography , enzyme
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 . However, the two forms of RII are markedly different in their (a) antigenic determinants, (b) cell and tissue distribution, and (c) subcellular localization. This suggests that 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 two proteins were mapped by reverse phase HPLC on a C 18 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 two‐dimensional mapping analyses revealed that several 32 P‐labeled tryptic fragments derived from autophosphorylated and photoaffinity‐labeled brain RII were separate and distinct from the 32 P‐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 suggest that the two cAMP‐binding proteins are different proteins rather than interconvertible forms of a single protein. The two‐dimensional maps further indicate that significant structural dissimilarities between brain RII and heart RII also occur within the functionally conserved autophosphorylation and cAMP‐binding domains.