Assay of cyclic AMP-dependent protein kinase activity in canine myocardium: effect of coronary artery ligation on the cytosolic enzyme.
A procedure for the determination of the activation state of cyclic AMP-dependent protein kinase (cAMP-PK) in the cytosolic fraction of dog myocardium was derived from existing assays of this enzyme in tissues, a new element being the absence of added sodium chloride throughout the procedure. About 2/3 of the total cAMP-PK in normal dog ventricular myocardium was found to be present in the cytosolic (supernatant) fraction, the remaining 1/3 being particle-bound. The isoenzyme profile in the cytosolic fraction as determined by DEAE-cellulose chromatography revealed the presence of nearly equal amounts of the type I and type II isoenzymes. This distribution was also evident in experiments in which the RI and RII regulatory subunits were photoaffinity labeled with 8-azido- [32P] cAMP. Ligation of the left circumflex coronary artery in anesthetized open-chest dogs kept under artificial respiration caused increases in the cAMP-PK activity ratio (-cAMP/+cAMP) from a control value of 0.11 to 0.26 and 0.22, respectively, in the ischemic and in a nonischemic area of the left ventricle. This effect was clearly evident 2 min after ligation and was still observable in the ischemic area after 20 min. It was associated with increases in cAMP levels and in the activity ratio (-5' -AMP/+5' -AMP) of glycogen phosphorylase in the ventricular tissues. Pretreatment of the dogs with (+/-)-propranolol (2 mg/kg) abolished the cAMP and cAMP-PK responses, while the phosphorylase activity ratio remained elevated.
D000332:Aerobiosis; D000818:Animals; D003327:Coronary Disease / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D006005:Phosphorylases / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
D007527:Isoenzymes; D011506:Proteins; D012965:Sodium Chloride; D011433:Propranolol; D006005:Phosphorylases; D011494:Protein Kinases