CAMP-DEPENDENT PROTEIN-KINASE AND ANOXIA SURVIVAL IN TURTLES - PURIFICATION AND PROPERTIES OF LIVER PKA

The catalytic subunit of turtle (Trachemys scripta elegans) liver cycl ic AMP-dependent protein kinase (PKAc) was purified to homogeneity wit h a final specific activity of 65,783 pmol phosphate transferred.min(- 1).mg protein(-1). Subunit molecular weight was 42-43 kDa as determine d by SDS-PAGE and Sephacryl S-300 chromatography. The isoelectric poin t 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. K-m values were 83 +/- 6.5 mu M for Mg.ATP and 11.7 +/- 0.5 mu M for kemptide and enzyme activity was strongly reduc ed by inhibitors of mammalian PKA (H-89, PKA-1) but not by inhibitors of other protein kinases. The enzyme was also inhibited by salts, espe cially fluoride salts (I-50 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 ab ove versus below 15 degrees C. Temperature effects may be important in suppressing PKA function, and therefore PKA-mediated responses, in vi vo to enhance anoxic survival time during winter hibernation under wat er. 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 accompanie d by a 60% increase cAMP levels; with longer anoxia (5 or 20 h) the pe rcentage of active PKA was suppressed to 2.1-3.7% of the total. Neithe r total nor active PKA changed in heart, brain, or white muscle during anoxia but the percent active decreased in anoxic red muscle. Anoxia- induced changes in PKA in liver support the enhanced glucogenesis need ed for fermentative energy production but the limited extent of PKA ac tivation (effects reversed by 5 h) in liver and the lack of change in most other organs is consistent with the primary defense for anoxia su rvival in turtles, metabolic rate depression.