Purification and characterization of the catalytic subunit of adenosine 3':5'-cyclic monophosphate-dependent protein kinase from bovine liver.
1. The catalytic subunit of bovine liver cyclic AMP-dependent protein kinase (EC2.7.1.37) was purified essentially by the method of Reimann & Corbin [(1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 1384]. 2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation-velocity centrifugation and sedimentation-equilibrium centrifugation showed that the catalytic subunit was monodisperse. Polyacrylamide-gel isoelectric-focusing electrophoresis revealed the presence of at least three isoenzyme forms of catalytic subunit activity with slightly different pI values (6.72, 7.04 and 7.35). 3. Physical properties of the catalytic subunit were determined by several different methods. It had mol.wt. 39000-42000, Stokes radium 2.73-3.08 nm, so20.w 3.14S, f/fo 1.19-1.23 and, assuming a prolate ellipsoid, axial ration 4-5. 4. Amino acid analysis was performed on the catalytic subunit. It had one cysteine residue/molecule which was essential for activity. Inhibition by thiol-specific reagents was partially prevented by the presence of ATP-Mg2+. 5. The circular-dichroic spectrum showed the catalytic subunit contained 29% alpha-helical form, 18% beta-form and 53% aperiodic form. Near-u.v. circular dichroism showed the presence of aromatic residues whose equivalent molar ellipticity was greatly altered by the addition of ATP-Mg2+. 6. Kinetic experiments showed that the catalytic subunit had an apparent Km for ATP of 7 muM. 5'-Adenylyl imidodiphosphate inhibitied competitively with ATP with a Ki of 60 muM. The kinetic plot for histone (Sigma, type II-A) was biphasic showing 'high'-and 'low'-Km segments. Under assay conditions the specific activity of the catalytic subunit was 3 X 10(6) units/mg of protein. Of various metal ions tested, the catalytic subunit was most active with Mg2+.7. When assayed with histone (Sigma, type II-A) as substrate, the activity of the catalytic subunit was increased by non-ionic detergents or urea. No such activation was observed with casein as substrate.
D000255:Adenosine Triphosphate; D000375:Aging; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002942:Circular Dichroism; D000242:Cyclic AMP; D004591:Electrophoresis, Polyacrylamide Gel; D007460:Iodoacetamide; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008670:Metals; D008970:Molecular Weight; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification*; D014461:Ultracentrifugation; D014508:Urea
D000596:Amino Acids; D008670:Metals; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D014508:Urea; D000242:Cyclic AMP; D011494:Protein Kinases; D007460:Iodoacetamide