Modulation of mammalian cardiac AMP deaminase by protein kinase C-mediated phosphorylation.
Using AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) purified from rabbit left-ventricular heart tissue, we report direct investigation of the potential for cardiac AMP deaminase activity to be regulated by kinase-mediated phosphorylation. Rabbit heart AMP deaminase served as a substrate for Ca2+/phospholipid-dependent protein kinase (protein kinase C; PKC) exclusively; no other mammalian protein kinase phosphorylated the enzyme. PKC-dependent AMP deaminase phosphorylation was rapid, linear with respect to time and the concentrations of PKC and AMP deaminase in the reaction, and inhibitable by staurosporine. Upon phosphorylation, the apparent Km of cardiac AMP deaminase decreased from 5.6 mM to 1.2 mM, without effect on the Vmax. Whether phosphorylated or not, rabbit heart AMP deaminase was inhibited by 1.0 mM GTP, which decreased the Vmax. by approximately 50% in each case. PKC-dependent phosphorylation of cardiac AMP deaminase did not alter the enzyme's allosterism toward millimolar ATP or ADP: both nucleotides at 1.0 mM concentration decreased the apparent Km to approximately 0.5 mM. Treatment of cardiac phospho-AMP deaminase with either the protein phosphatase calcineurin or alkaline phosphatase generated a dephosphorylated form which displayed molecular and kinetic properties identical with those of the originally isolated enzyme. These data raise the possibility that a phosphorylation-dephosphorylation mechanism may regulate flux through AMP deaminase in the heart under pathological conditions, such as myocardial ischaemia, characterized by PKC activation and adenylate depletion.
Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit, NJ 07901.
J K
JK
Thakkar
D R
DR
Janero
C
C
Yarwood
H M
HM
Sharif
D000659:AMP Deaminase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D019311:Staurosporine
D000470:Alkaloids; D002148:Calmodulin-Binding Proteins; D006160:Guanosine Triphosphate; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000659:AMP Deaminase; D019311:Staurosporine