Isolation of an inactive bovine heart cAMP-dependent protein kinase holoenzyme containing bound cAMP

Journal Article · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5023076

Cobb, C E; Beth, A H; Corbin, J D

Bovine heart Type II cAMP-dependent protein kinase (PK) was purified to homogeneity as determined by SDS-PAGE. The purification steps were DEAE-cellulose, ammonium sulfate precipitation, phenyl-Sepharose, alumina C-..gamma.., and HPLC-DEAE. The last step resolved two distinct peaks of cAMP dependent kinase activity (activity ratios = 0.05 - 0.10) eluting at approximately 250 (Peak 1) and 275 (Peak 2) mM NaCl. When subjected to HPLC-gel permeation they had the same Stoke's radii. Linear sucrose gradients gave S/sub 20,w/ values of 7.73 S and 7.25 S for Peaks 1 and 2, respectively. It was determined by integrating the areas of scanned SDS-PAGE bands that regulatory and catalytic subunits were present in equimolar amounts in both Peaks 1 and 2. The ratios of equilibrium (/sup 3/H) cAMP binding to kinase activity for the two peaks were the same. cAMP was present in trace amounts in Peak 1 but in stoichiometric amounts in Peak 2 (between 25 and 75 % saturation of cAMP binding sites). 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. Enhanced positive cooperativity in nonequilibrium (/sup 3/H) cAMP binding and in kinase activation (as indicated by a larger Hill coefficient) of Peak 2 relative to Peak 1 also supported the presence of cAMP in Peak 2. In conclusion, two forms of inactive PK have been isolated, one of which is a ternary complex of PK holoenzyme and cAMP. This complex could represent a cellular form of the enzyme which is primed for activation.

• Research Organization: Vanderbilt Univ., Nashville, TN
• OSTI ID: 5023076
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

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