Use of High Performance Liquid Chromatography in Characterizing Nucleotide Binding Sites and Antigenic Determinants in cAMP-Dependent Protein Kinase
cAMP-dependent protein kinase is a multisubunit protein whose activity and quaternary structure are both modulated by cAMP (1,2). In its native holoenzyme form, the enzyme is an inactive tetramer containing two regulatory (R) and two catalytic (C) subunits. In the presence of cAMP, the holoenzyme dissociates into a regulatory subunit dimer (R2) and two monomeric catalytic subunits with each R2 binding 4 molecules of cAMP (3,4). Because of the multiple aggregation states that serve a regulatory role and because of several substrate and modulator binding sites, this enzyme provides an excellent model for investigating protein-protein interactions, nucleotide-protein interactions, and enzyme regulation. We have utilized high performance liquid chromatography (HPLC) in conjunction with solid phase sequencing to characterize a variety of specific sites on both subunits of the kinase purified from porcine cardiac muscle.
KeywordsHigh Performance Liquid Chromato Antigenic Site Limited Proteolysis High Performance Liquid Chromato High Performance Liquid Chromato
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