cAMP-dependent protein kinase and anoxia survival in turtles: purification and properties of liver PKA.
The catalytic subunit of turtle (Trachemys scripta elegans) liver cyclic AMP-dependent protein kinase (PKAc) was purified to homogeneity with a final specific activity of 65,783 pmol phosphate transferred.min-1.mg protein-1. Subunit molecular weight was 42-43 kDa as determined by SDS-PAGE and Sephacryl S-300 chromatography. The isolectric point was pH 6.41 +/- 0.02. Turtle liver PKAc showed highest activity with kemptide as its substrate; activity with other artificial substrates, histone IIA and protamine, was only 21 and 11%, respectively, of the activity with kemptide. Km values were 83 +/- 6.5 microM for Mg.ATP and 11.7 +/- 0.5 microM for kemptide and enzyme activity was strongly reduced by inhibitors of mammalian PKA (H-89, PKA-1) but not by inhibitors of other protein kinases. The enzyme was also inhibited by salts, especially fluoride salts (I50 about 30 mM), and showed a sharp break in the Arrhenius plot (0-45 degrees C) with activation energy increasing by 4-fold from 27.9 +/- 1.85 to 115 +/- 2.5 kJ/mol for temperatures above versus below 15 degrees C. Temperature effects may be important in suppressing PKA function, and therefore PKA-mediated responses, in vivo to enhance anoxic survival time during winter hibernation under water. Analysis of the effects of in vivo anoxia exposure at 7 degrees C on PKA in turtle organs showed a rapid 2.3-fold increase in the amount of active enzyme in liver within 1 h of anoxic submergence accompanied by a 60% increase cAMP levels; with longer anoxia (5 or 20 h) the percentage of active PKA was suppressed to 2.1-3.7% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)
Mehrani
H
H
Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
Storey
K B
KB
D000818:Animals; D001921:Brain / Q000378:metabolism; D002845:Chromatography; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D000860:Hypoxia / Q000378:metabolism*; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D013997:Time Factors; D014426:Turtles
D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases