Journal of Comparative Physiology B

, Volume 157, Issue 6, pp 813–820 | Cite as

Role of covalent modification in the control of glycolytic enzymes in response to environmental anoxia in goldfish

  • M. S. Rahman
  • K. B. Storey
Article

Summary

The effects of environmental anoxia (24 h at 7°C in N2/CO bubbled water) on the maximal activities, selected kinetic properties, and isoelectric points of phosphofructokinase and pyruvate kinase were measured in eight tissues of the goldfish,Carassius auratus, in order to evaluate the role of possible covalent modification of enzymes in glycolytic rate control and metabolic depression during facultative anaerobiosis. Both enzymes showed modified kinetic properties as a result of anoxia in liver, kidney, brain, spleen, gill, and heart. Effects of anoxia on properties of pyruvate kinase included reducedVmax, increased S0.5 for phosphoenolpyruvate, increasedKa for fructose-1,6-bisphosphate, and strongly reduced I50 for alanine; all these effects are consistent with an anoxia-induced phosphorylation of pyruvate kinase to produce a less active enzyme form. Anoxia-induced alterations in phosphofructokinase kinetics included tissue-specific changes in S0.5 for fructose-6-phosphate, Hill coefficient,Ka values for fructose-2,6-bisphosphate, AMP, and NH4+, and I50 values for ATP and citrate, the direction of changes being generally consistent with the production of a less active enzyme form in the anoxic tissue. Enzymes from aerobic versus anoxic skeletal muscle (both red and white) did not differ in kinetic properties but anoxic enzyme forms had significantly different pI values than the corresponding aerobic forms. Enzyme phosphorylation-dephosphorylation as the basis of the anoxia-induced changes in the kinetic properties of PFK and PK was further tested in liver: treatment of the aerobic forms of both enzymes with cAMP dependent protein kinase altered enzyme kinetic properties to those typical of the anoxic enzymes while alkaline phosphatase treatment of the anoxic enzyme forms had the opposite effect. The data provide strong evidence that coordinated glycolytic rate control, as part of an overall metabolic rate depression during anoxia, is mediated via anoxia-induced covalent modification of regulatory enzymes.

Abbreviations

cAMP

cyclic 3′5′ adenosine monophosphate

F16P2

fructose-1,6-bisphosphate

F26P2

fructose-2,6-bisphosphate

F6P

fructose-6-phosphate

PEP

phosphoenolpyruvate

PFK

phosphofructokinase (E.C. 2.7.1.11)

PK

pyruvate kinase (E.C. 2.7.1.40)

PMSF

phenylmethylsulfonyl fluoride

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References

  1. Andersen J (1975) Anaerobic resistance ofCarassius auratus (L.). PhD thesis. Australian National University, CanberraGoogle Scholar
  2. Engstrom L (1978) The regulation of liver pyruvate kinase by phosphorylation-dephosphorylation. In: Horecker B, Stadtman E (eds) Current topics in cellular regulation, vol 13. Academic Press, New York, pp 29–51Google Scholar
  3. Foe LG, Kemp RG (1982) Properties of phospho and dephospho forms of muscle phosphofructokinase. J Biol Chem 257:6368–6372Google Scholar
  4. Foe LG, Kemp RG (1984) Isozyme composition and phosphorylation of brain phosphofructokinase. Arch Biochem Biophys 228:503–511Google Scholar
  5. Luther MA, Lee JC (1986) The role of phosphorylation in the interaction of rabbit muscle phosphofructokinase with F-actin. J Biol Chem 261:1753–1759Google Scholar
  6. Marie O, Buc H, Simon M-P, Kahn A (1980) Phosphorylation of human erythrocyte pyruvate kinase by soluble cyclic-AMP-dependent protein kinase. Eur J Biochem 108:251–260Google Scholar
  7. Narabayashi H, Randolph Lawson JW, Uyeda K (1985) Regulation of phosphofructokinase in perfused rat heart. Requirement for fructose-2,6-bisphosphate and a covalent modification. J Biol Chem 260:9750–9758Google Scholar
  8. Plaxton WC, Storey KB (1984a) Purification and properties of aerobic and anoxic forms of pyruvate kinase from red muscle tissue of the channeled whelk,Busycotypus canaliculatum. Eur J Biochem 143:257–265Google Scholar
  9. Plaxton WC, Storey KB (1984b) Phosphorylation in vivo of red muscle pyruvate kinase from the channeled whelk,Busycotypus canaliculatum: enzyme modification in response to environmental anoxia. Eur J Biochem 143:267–272Google Scholar
  10. Plaxton WC, Storey KB (1985) Tissue specific isozymes of pyruvate kinase in the channeled whelk,Busycotypus canaliculatum: enzyme modification in response to environmental anoxia. J Comp Physiol 155:291–296Google Scholar
  11. Sakakibara R, Uyeda K (1983) Differences in the allosteric properties of pure low and high phosphate forms of phosphofructokinase from rat liver. J Biol Chem 258:8656–8662Google Scholar
  12. Shoubridge EA, Hochachka PW (1981) The origin and significance of metabolic carbon dioxide production in the anoxic goldfish. Mol Physiol 1:315–338Google Scholar
  13. Shoubridge EA, Hochachka PW (1983) The integration and control of metabolism in the anoxic goldfish. Mol Physiol 4:165–195Google Scholar
  14. Storey KB (1984) Phosphofructokinase from foot muscle of the whelk,Busycotypus canaliculatum: Evidence for covalent modification of the enzyme during anaerobiosis. Arch Biochem Biophys 235:665–672Google Scholar
  15. Storey KB (1985) A re-evaluation of the Pasteur effect: new mechanisms in anaerobic metabolism. Mol Physiol 8:439–461Google Scholar
  16. Storey KB (1987a) Suspended animation: The molecular basis of metabolic depression. Can J Zool (in press)Google Scholar
  17. Storey KB (1987b) Tissue specific controls on carbohydrate catabolism during anoxia in goldfish. Physiol Zool (in press)Google Scholar
  18. Van den Thillart G, Verbeek R (1982) Substrates for anaerobic CO2 production by the goldfish,Carassius auratus (L.): decarboxylation of14C-labelled metabolites. J Comp Physiol 149:75–81Google Scholar
  19. Van den Thillart G, Van Berge-Henegouwen M, Kesbeke F (1983) Anaerobic metabolism of goldfish,Carassius auratus (L.): ethanol and CO2 excretion rates and anoxia tolerance at 20, 10 and 5°C. Comp Biochem Physiol 76A:295–300Google Scholar
  20. Van den Thillart G, Kesbeke F, Van Waarde A (1976) Influence of anoxia on the energy metabolism of the goldfish,Carassius auratus L. Comp Biochem Physiol 59A:329–336Google Scholar
  21. Walker RM, Johansen PH (1977) Anaerobic metabolism in goldfish (Carassius auratus). Can J Zool 55:304–311Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • M. S. Rahman
    • 1
  • K. B. Storey
    • 1
  1. 1.Institute of Biochemistry and Department of BiologyCarleton UniversityOttawaCanada

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