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Protein kinase involvement in land snail aestivation and anoxia: Protein kinase A kinetic properties and changes in second messenger compounds during depressed metabolism

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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 affecting 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 anoxia exposure and aestivation in foot. Cyclic AMP concentrations in hepatopancreas transiently decreased with the lowest concentration 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 IP3 concentrations were significantly lower in 24 h anoxic snails and foot IP3 concentrations were significantly lower in 24 h aestivating snails. Kinetic characterization of purified PKA catalytic subunit was also performed. Snail PKA catalytic subunit had an absolute requirement for Mg2+ ion but was inhibited at Mg2+ concentrations above 0.5 mM. Increasing concentrations of neutral salts and phosphate also inhibited activity although the inhibition by phosphate appeared to be specific since the inhibition constant (I50 = 39 mM) was much lower than that of the neutral salts (I50 ≈ 240 mM). 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 Q10 value of 2.3. The relationship between these results and temporal control of enzyme phosphorylation is discussed.

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Abbreviations

CAMP:

adenosine 3′:5′-cyclic monophosphate

cGMP-guanosine:

3′:5′-cyclic monophosphate

H-89N:

[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCI

IP3 :

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 subunit

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

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Author notes

  1. Stephen P. J. Brooks

    Present address: Nutrition Research Division, Health Protection Branch, Health Canada, 3W Banting Research Centre, Tunney's Pasture, 2203C Ottawa, KIA OL2, Ontario, Canada

Authors and Affiliations

  1. Department of Biology and Institute of Biochemistry, Carleton University, KIS 5B6, Ottawa Ontario, Canada

    Stephen P. J. Brooks & Kenneth B. Storey

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Brooks, S.P.J., Storey, K.B. Protein kinase involvement in land snail aestivation and anoxia: Protein kinase A kinetic properties and changes in second messenger compounds during depressed metabolism. Mol Cell Biochem 156, 153–161 (1996). https://doi.org/10.1007/BF00426338

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