Cyclic AMP-dependent protein kinase: role in anoxia and freezing toleranceof the marine periwinkle Littorina littorea

A key regulatory mechanism underlying the switch between aerobic and anaero bic metabolism amongst anoxia-tolerant marine molluscs is reversible protei n phosphorylation. To assess the role of cAMP-dependent protein kinase (PKA ) in aerobic-anaerobic transitions, the effects of anoxia on the activity a nd subcellular distribution of PKA were assessed in foot and hepatopancreas of the marine periwinkle, Littorina littorea. Exposure to N-2 gas at 5 OC caused a rapid decline in the percentage of total enzyme present as the fre e catalytic subunit (PKAc) in both tissues; the percentage of PKAc fell fro m similar to 30% in controls to 3% after 1 h anoxia and remained low over 7 2 h. Total PKA also fell by 30% after 72 h anoxia in hepatopancreas but reb ounded during aerobic recovery. Freezing at -8 degrees C elicited parallel results for both percentage of PKAc and total PKA, suggesting that PKA resp onses to freezing were stimulated by the ischemia that develops when hemoly mph freezes. Anoxia also led to a shift in PKA subcellular distribution in hepatopancreas (but not in foot), the percentage of total PKA activity asso ciated with the nuclear fraction dropping from 25% in controls to 8% in 12 h anoxic snails with opposite changes in the cytosolic fraction. The cataly tic subunit (PKAc) of foot PKA was purified to a final specific activity of 63.5 nmol phosphate transferred per minute per milligram protein. Enzyme p roperties included a molecular weight of 33 to 35 kDa, an activation energy from Arrhenius plots of 65.1 +/- 4.8 kJ mol(-1), and substrate affinity co nstants of 151 +/- 6 mu M for the phosphate acceptor, Kemptide, and 72 +/- 9 mu M for Mg.ATP. Activity was strongly reduced by mammalian PKA inhibitor s (H-89, PKA-I), by neutral chloride salts (I-50 values 165 to 210 mM) and by NaF (I-50 62 mM). Reduced PKA activity under anoxic or freezing conditio ns would facilitate the observed suppression of the activities of numerous enzymes that are typically PKA-activated and thereby contribute to the over all anoxia-induced metabolic rate depression.