Purified pyruvate kinases type M2 from unfertilized hen's egg are substrates of protein kinase C.
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 mumol X min-1 X mg-1. It was saturated half-maximally with phosphoenolpyruvate at KPPrv0.5 = 0.36 mM phosphoenolpyruvate and was activity by fructose 1,6-bisphosphate and L-serine at suboptimal substrate concentrations. After 11 000-fold purification to a specific enzymatic activity of 60 mumol X min-1 X mg-1, the pyruvate kinase isoenzymes type M2 (KPPrv0.5 = 0.32 mM) and M1 (KPPrv0.5 = 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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D004530:Egg Yolk / Q000032:analysis; D005260:Female; D005306:Fertilization; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007526:Isoelectric Point; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008970:Molecular Weight; D010063:Ovum / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014817:Vitelline Membrane / Q000201:enzymology
D000596:Amino Acids; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011770:Pyruvate Kinase; D011493:Protein Kinase C