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Isolation and characterization of cAMP-free and cAMP-bound forms of bovine heart type II cAMP-dependent protein kinase

Thesis/Dissertation ·
OSTI ID:5243595
Bovine heart type II cAMP-dependent protein kinase holoenzyme (cAMP-PK) was purified to homogeneity as determined by denaturing SDS-PAGE. An HPLC-DEAE purification step resolved two distinct peaks of cAMP-dependent kinase activity, which were designated Peak 1 and Peak 2 based on their order of elution. They had the same Stoke's radii and had very similar sedimentation coefficients. As determined by densitometric scanning of SDS-PAGE brands, by their mobility on denaturing PAGE, and by the ratios of equilibrium (/sup 3/H) cAMP binding to maximal kinase activity, the subunit stoichiometry of the two peaks was the same. In a cAMP assay it was found that Peak 1 holoenzyme was cAMP-free, but half of the Peak 2 holoenzyme cAMP binding sites contained cAMP. Dissociation assays indicated that the cAMP was equally distributed in binding Site 1 and Site 2 of Peak 2. Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP, and Peak 2 could be partially converted to Peak 1 by aging. The interconvertibility of the two holoenzyme peaks strongly suggested that the difference between the two peaks was caused by the presence of cAMP in Peak 2.
Research Organization:
Vanderbilt Univ., Nashville, TN (USA)
OSTI ID:
5243595
Country of Publication:
United States
Language:
English

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Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMP
ANIMALS
BIOCHEMICAL REACTION KINETICS
BODY
CARDIOVASCULAR SYSTEM
CATTLE
DOMESTIC ANIMALS
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
FRACTIONATION
HEART
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
MAMMALS
MEMBRANE PROTEINS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEINS
REACTION KINETICS
RECEPTORS
RUMINANTS
SEPARATION PROCESSES
STOICHIOMETRY
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
VERTEBRATES