Abstract
The effects of anoxia (N2 atmosphere at 5 °C) or freezing (at-8 °C) exposure in vivo on the activities of five enzymes of carbohydrate metabolism were assessed in foot muscle and hepatopancreases of the marine periwinkle Littorina littorea. Changes in glycogen phosphorylase, glycogen synthetase, pyruvate kinase and pyruvate dehydrogenase under either stress were generally consistent with covalent modification of the enzymes to decrease enzyme activity and/or convert the enzyme to a less active form. However, no evidence for a similar covalent modification of phosphofructokinase was found. The metabolic effects of freezing and anoxia were generally similar, suggesting that a primary contributor to freezing survival is the implementation of anaerobic metabolism and metabolic arrest mechanisms that also promote anoxia survival in marine molluses. However, in hepatopancreas phosphorylase was activated and pyruvate kinase remained in two enzyme forms in freezing-exposed snails, contrary to the results for anoxic animals. Ion exchange chromatography on DE-52 Sephadex revealed the presence of two forms of pyruvate kinase in both tissues of control L. littorea, eluting at 30–50 mmol·1-1 KCl (peak I) or 90–110 mmol·1-1 KCl (peak II). Anoxia exposure converted pyruvate kinase in both tissues to the peak I form, as did freezing for foot muscle pyruvate kinase. Kinetic analysis showed that peak I pyruvate kinase had lower affinities for substrates, phosphoenolpyruvate and ADP, and was very strongly inhibited by l-alanine compared with the peak II enzyme. Peak I pyruvate kinase had an I 50 value for l-alanine of 0.38 mmol·1-1, whereas peak II pyruvate kinase was unaffected by l-alanine evenat 40 mmol·1-1. In vitro incubation of extracts from control foot muscle under conditions promoting phosphorylation or dephosphorylation identified the peak I and II forms as the low and high phosphate forms, respectively. This result for L. littorea pyruvate kinase was highly unusual and contrary to the typical effect of anoxia on pyruvate kinase in marine molluscs which is to stimulate the phosphorylation of pyruvate kinase and, thereby, convert the enzyme to a less active form.
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Abbreviations
- AABS :
-
p-(p-aminophenylazo)benzene sulphonic acid
- F2, 6P :
-
fructose-2,6-bisphosphate
- F6P :
-
fructose-6-phosphate
- G6P :
-
glucose-6-phosphate
- GP :
-
glycogen phosphorylase
- GS :
-
glycogen synthase
- I 50 :
-
inhibitor concentration reducing enzyme velocity by 50%
- MR :
-
metabolic rate
- PDH :
-
pyruvate dehydrogenase
- PEP :
-
phosphoenopyruvate
- PFK :
-
phosphofructokinase
- PK :
-
pyruvate kinase
- SW :
-
sea water
- F a :
-
air temperature
- TCA :
-
trichloroacetic acid
- UDPG :
-
uridine-diphosphate glucose
- WW :
-
wet weight
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Communicated by L.C.-H. Wang
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Russell, E.L., Storey, K.B. Anoxia and freezing exposures stimulate covalent modification of enzymes of carbohydrate metabolism in Littorina littorea . J Comp Physiol B 165, 132–142 (1995). https://doi.org/10.1007/BF00301477
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DOI: https://doi.org/10.1007/BF00301477