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cAMP-dependent protein kinase and anoxia survival in turtles: Purification and properties of liver PKA

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Abstract

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 isoelectric 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 μM for Mg.ATP and 11.7±0.5 μM 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°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°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 ofin vivo anoxia exposure at 7°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. Neither 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 needed for fermentative energy production but the limited extent of PKA activation (effects reversed by 5 h) in liver and the lack of change in most other organs is consistent with the primary defense for anoxia survival in turtles, metabolic rate depression.

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Abbreviations

PKA:

cyclic AMP-dependent protein kinase

PKAc:

catalytic subunit of cyclic AMP-dependent protein kinase

PEG:

poly(ethyleneglycol)

cAMP:

cyclic, 3′,5′-adenosine monophosphate

SDS:

sodium dodecyl sulfate

I50 :

inhibitor concentration that lowers enzyme velocity by 50%

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Authors and Affiliations

  1. Institute of Biochemistry and Department of Chemistry, Carleton University, K1S 5B6, Ottawa, Ontario, Canada

    Hossein Mehrani & Kenneth B. Storey

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  1. Hossein Mehrani
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  2. Kenneth B. Storey
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Mehrani, H., Storey, K.B. cAMP-dependent protein kinase and anoxia survival in turtles: Purification and properties of liver PKA. Mol Cell Biochem 145, 81–88 (1995). https://doi.org/10.1007/BF00925717

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  • DOI: https://doi.org/10.1007/BF00925717

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