Pyruvate kinase from the earthworm Allolobophora calliginosa: modification of the enzyme during anaerobiosis

Abstract

The relative activity of pyruvate kinase from the body-wall muscle of the earthworm Allolobophora calliginosa was found to drop dramatically within 6 h of exposure to N2, whereas the opposite was observed during recovery. Two forms of pyruvate kinase (designated as peak I and peak II) were separated chromatographically on DEAE-cellulose and eluted at 50 and 150 mM of KCl, respectively. They displayed different kinetic behaviour with respect to substrate phosphoenolpyruvate; peak I exhibited Michaelis–Menten kinetics whereas peak II showed sigmoidal kinetics. The ratio of the enzyme units (peak I/peak II) decreased from 3.38 under normoxic conditions to 0.09 under anoxic conditions. In vitro incubation of the aerobic form of pyruvate kinase in the presence of ATP and Mg++ resulted in a reduction of the enzyme activity by 64%, suggesting the presence of an endogenous cyclic-nucleotide-independent protein kinase capable of phosphorylating pyruvate kinase. After in vitro incubation, alkaline phosphatase from E. coli increased the depressed activity of anaerobic pyruvate kinase, indicating that the enzyme molecule is phosphorylated in vivo during exposure to anoxia.