Regulation of glycolytic enzyme activity during chronic hypoxia by changes in rate-limiting enzyme content. Use of monoclonal antibodies to quantitate changes in pyruvate kinase content.

摘要

Monoclonal antibodies were prepared against pyruvate kinase (PyKi; ATP: pyruvate phosphotransferase, EC 2.7.1.40) and used to quantitate PyKi content in L2 lung cells and WI-38 fibroblasts cultivated under hypoxic and normoxic conditions. After 96 h of hypoxic cultivation, PyKi activity was significantly increased in both cell types (L2: normoxia [Po2 = 142 torr], 0.11 +/- 0.01 [SD]; hypoxia [Po2 = 14 torr], 0.25 +/- 0.04 U/microgram DNA, P < 0.01). PyKi content increased proportionately in both cell lines (L2: normoxia, 0.44 +/- 0.13; hypoxia, 0.94 +/- 0.13 microgram enzyme protein/microgram DNA). Specific activity was not significantly different after 96 h (L2: normoxia, 261 +/- 11; hypoxia, 261 +/- 14 U/mg enzyme protein). These results indicate that regulation of glycolysis during chronic hypoxia occurs at the level of enzyme content. Chronic O2 depletion leads to either an increased rate of biosynthesis or a decreased rate of biodegradation of PyKi, causing augmented glycolytic capacity. Monoclonal antibodies provide a highly specific, convenient approach to charcterizing enzymes, as well as quantitating cellular enzyme content.