The response of Crassostrea gigas to prolonged
hypoxia was investigated for the first time by analyzing the metabolic branch point formed by
pyruvate kinase (PK) and hosphoenolpyruvate carboxykinase (PEPCK). PK and PEPCK cDNAs were cloned and sequenced. The main functional domains of the PK sequence, such as the binding sites for
ADP/
ATP and
phosphoenolpyruvate (PEP), were identified whereas the PEPCK sequence showed the specific domain to bind PEP in addition to the kinase-1 and kinase-2 motifs to bind
guanosine triphosphate (
GTP) and Mg(2+), specific for all PEPCKs. A C-terminal extension was detected for the first time in eukaryota PK. Separation of mitochondrial and cytosolic fraction showed that more than 92% of the PEPCK
enzyme activity was cytosolic in gills, digestive gland, mantle and muscle. PK and PEPCK mRNAs and
enzyme activities have been measured in muscle during prolonged
hypoxia for 20 days. Adaptation of PK in hypoxic muscle at transcriptional level occurred lately by decreasing significantly the PK
mRNA level at day 20 while PK
enzyme activity was inhibited by the high content of
alanine. The PEPCK
mRNA ratio in hypoxic muscle significantly increased at day 10 simultaneously to the PEPCK
enzyme activity.
Succinate accumulation observed at day 10 and day 20 confirmed the anaerobic pathway of muscle metabolism in oyster subjected to
hypoxia. Regulation of C. gigas PEPCK in muscle occurred at gene transcription level while PK was first regulated at
enzyme level with
alanine as allosteric inhibitor, and then at molecular level under a fast effect of
hypoxia.