Purified pyruvate kinases type M2 from unfertilized hen's egg are substrates of protein kinase C

Abstract

To characterize pyruvate kinase isoenzymes from cells with the capability to proliferate, this enzyme was purified from yolk and vitelline membrane of unfertilized hen's egg. Pyruvate kinase type M₂ from vitelline membrane was obtained in a homogeneous form after a 1150-fold purification to a specific enzymatic activity of 450 μmol · min⁻¹· mg⁻¹. It was saturated half-maximally with phosphoenolpyruvate at K^PPrv_0.5= 0.36 mM phosphoenolpyruvate and was activated by fructose 1,6-bisphosphate and L-serine at suboptimal substrate concentrations. After 11000-fold purification to a specific enzymatic activity of 60 μmol min⁻¹· mg⁻¹, the pyruvate kinase isoenzymes type M₂ (K^PPrv_0.5= 0.32 mM) and M₁ (K^PPrv_0.5= 0.04 mM) were obtained from the yolk substance. Kinetic differences were noted between the pyruvate kinase type-M₂ isoenzymes from vitelline membrane and yolk. A comparison of the amino acid composition of the purified pyruvate kinase isoenzymes from hen's egg revealed that all isoenzymes were related to pyruvate kinase type M₁ from chicken breast muscle. The M₂-type isoenzyme from vitelline membrane was related to the M₂-type isoenzyme from chicken tumors, but was not related to the M₂-type pyruvate kinase from chicken lung or liver. Protein kinase C from chicken oviduct phosphorylated in vitro both pyruvate kinase M₂ isoenzymes from the unfertilized hen's egg preferably at serine and less at threonine residues. Pyruvate kinase type M₁ from egg yolk was a weak substrate of protein kinase C. An activation of pyruvate kinase type M₂ from vitelline membrane was observed at suboptimal concentrations of phosphoenolpyruvate under the conditions of phosphorylation, in the presence of phosphatidylserine.