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

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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 reducedV max, increased S0.5 for phosphoenolpyruvate, increasedK a 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,K a values for fructose-2,6-bisphosphate, AMP, and NH +4 , 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.

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Abbreviations

cAMP :

cyclic 3′5′ adenosine monophosphate

F16P 2 :

fructose-1,6-bisphosphate

F26P 2 :

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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Rahman, M.S., Storey, K.B. Role of covalent modification in the control of glycolytic enzymes in response to environmental anoxia in goldfish. J Comp Physiol B 157, 813–820 (1988). https://doi.org/10.1007/BF00691013

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  • DOI: https://doi.org/10.1007/BF00691013

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