PROTEIN-KINASE INVOLVEMENT IN LAND SNAIL ESTIVATION AND ANOXIA - PROTEIN-KINASE-A KINETIC-PROPERTIES AND CHANGES IN 2ND MESSENGER COMPOUNDSDURING DEPRESSED METABOLISM

In response to environmental stress (low water, low oxygen) snails sha rply suppress their metabolic rate, a process that is coordinated at t he molecular level by reversible protein phosphorylation of key enzyme s and functional proteins. Factors affecting protein kinase activity a re, therefore, critical to metabolic suppression. Changes in the conce ntration of protein kinase second messenger compounds were followed ov er the first 24 h of aestivation and anoxia exposure in the terrestria l snail Otala lactea (Muller) (Pulmonata, Helicidae). The results show ed declining concentrations of cyclic AMP over the first 24 h of anoxi a exposure and aestivation in foot. Cyclic AMP concentrations in hepat opancreas transiently decreased with the lowest concentration observed at 4 h in both anoxic and aestivating animals. A transient increase i n foot muscle cyclic GMP concentrations was apparent 4 h after the sta rt of aestivation whereas a slow, steady increase was seen in anoxic f oot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentratio ns decreased transiently during anoxia exposure and aestivation. Hepat opancreas IP3 concentrations were significantly lower in 24 h anoxic s nails and foot IP3 concentrations were significantly lower in 24 h aes tivating snails. Kinetic characterization of purified PKA catalytic su bunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg2+ ion but was inhibited at Mg2+ concentrations abov e 0.5 mM. Increasing concentrations of neutral salts and phosphate als o inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I-50 = 39 mM) was much lowe r than that of the neutral salts (I-50 approximate to 240 mM). The enz yme exhibited a broad pH optimum between pH 6.5-8.5. Arrhenius plots g ave an activation energy of 13.3 kcal/mol corresponding to a Q(10) val ue of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.