ANOXIA AND FREEZING EXPOSURES STIMULATE COVALENT MODIFICATION OF ENZYMES OF CARBOHYDRATE-METABOLISM IN LITTORINA-LITTOREA

The effects of anoxia (N-2 atmosphere at 5 degrees C) or freezing (at -8 degrees C) exposure in vivo on the activities of five enzymes of ca rbohydrate metabolism were assessed in foot muscle and hepatopancreas of the marine periwinkle Littorina littorea. Changes in glycogen phosp horylase, glycogen synthetase, pyruvate kinase and pyruvate dehydrogen ase under either stress were generally consistent with covalent modifi cation of the enzymes to decrease enzyme activity and/or convert the e nzyme to a less active form. However, no evidence for a similar covale nt modification of phosphofructokinase was found. The metabolic effect s of freezing and anoxia were generally similar, suggesting that a pri mary contributor to freezing survival is the implementation of anaerob ic metabolism and metabolic arrest mechanisms that also promote anoxia survival in marine molluscs. However, in hepatopancreas phosphorylase was activated and pyruvate kinase remained in two enzyme forms in fre ezing-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, elut ing at 30-50 mmol . l(-1) KCl (peak I) or 90-110 mmol . l(-1) KCl (pea k II). Anoxia exposure converted pyruvate kinase in both tissues to th e peak I form, as did freezing for foot muscle pyruvate kinase. Kineti c analysis showed that peak I pyruvate kinase had lower affinities for substrates, phosphoenolpyruvate and ADP, and was very strongly inhibi ted by L-alanine compared with the peak II enzyme. Peak I pyruvate kin ase had an I,, value for L-alanine of 0.38 mmol . l(-1), whereas peak II pyruvate kinase was unaffected by L-alanine evenat 40 mmol . l(-1). In vitro incubation of extracts from control foot muscle under condit ions promoting phosphorylation or dephosphorylation identified the pea k I and II forms as the low and high phosphate forms, respectively. Th is result for L. littorea pyruvate kinase was highly unusual and contr ary to the typical effect of anoxia on pyruvate kinase in marine mollu scs which is to stimulate the phosphorylation of pyruvate kinase and, thereby, convert the enzyme to a less active form.