Molecular and CellularBiochemistry 156:153-161, 1996. © 1996 Kluwer AcademicPublishers. Printedin the Netherlands. Protein kinase involvement in land snail aestivation and anoxia: Protein kinase A kinetic properties and changes in second messenger compounds during depressed metabolism Stephen RJ. Brooks 1 and Kenneth B. Storey Department of Biology and Institute of Biochemistry, Carleton University, Ottawa Ontario K1S 5B6, Canada Received 10 August 1995; accepted 17 November 1995 Abstract In response to environmental stress (low water, low oxygen) snails sharply suppress their metabolic rate, a process that is coordinated at the molecular level by reversible protein phosphorylation of key enzymes and functional proteins. Factors af- fecting protein kinase activity are, therefore, critical to metabolic suppression. Changes in the concentration of protein kinase second messenger compounds were followed over the first 24 h of aestivation and anoxia exposure in the terrestrial snail Otala lactea (Muller) (Pulmonata, Helicidae). The results showed declining concentrations of cyclic AMP over the first 24 h of an- oxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest con- centration observed at 4 h in both anoxic and aestivating animals. A transient increase in foot muscle cyclic GMP concentrations was apparent 4 h after the start of aestivation whereas a slow, steady increase was seen in anoxic foot muscle. Foot muscle 1,4,5-inositol triphosphate (IP3) concentrations decreased transiently during anoxia exposure and aestivation. Hepatopancreas IP 3 concentrations were significantly lower in 24 h anoxic snails and foot IP 3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization &purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg 2+ ion but was inhibited at Mg z+concentrations above 0.5 raM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (150= 39 raM) was much lower than that of the neutral salts (150~ 240 raM). The enzyme exhibited a broad pH optimum between pH 6.5-8.5. Arrhenius plots gave an activation energy of 13.3 kcal/mol corresponding to a Qt0 value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed. (Mol Cell Biochem 156: 153-161, 1996) Key words. protein kinase A, aestivation, anoxia, protein kinase second messengers Abbreviations: cAMP- adenosine 3':5'-cyclic monophosphate; cGMP- guanosine 3':5'-cyclic monophosphate; H-89- N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide,2HCI, IP 3- D-myo-inositol 1,4,5-triphosphate; I50-the concentration of inhibitor required to reduce the velocity to one half its original value; PKA - cAMP dependent protein kinase; PKAc- PKA catalytic snbunit; PKA-I- PKA inhibitor protein, PKC - calcium and phospholipid-dependent protein kinase, PKC-I - PKC inhibitor protein; PKG - cGMP dependent protein kinase; mU- nmol of phosphate transferred per minute ~Present address. Nutrition ResearchDivision, HealthProtectionBranch, HealthCanada, 3W Banting ResearchCentre, Tunney's Pasture, 2203C, Ottawa, Ontario, K1A 0L2, Canada Address for offprints." K.B. Storey,Depam-nent of Biology, Carleton University,Ottawa Ontario K1S 5B6, Canada