Purification and properties of pig heart pyruvate kinase.
Using essentially a two-step procedure involving phosphocellulose column chromatography followed by gel filtration on Sephadex G200, pig heart pyruvate kinase (PH PyK) was purified 267-fold to at least 97% purity. PH PyK co-sedimented with rabbit muscle PyK during sucrose density ultracentrifugation yielding an S20,w of 10 and a corresponding molecular weight of about 237,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a subunit molecular weight of approximately 59,000, suggesting that native PH PyK exists as a tetramer. The isoelectric point (pI) was determined to be 8.2, and the pH optimum (pHo) for the forward reaction is 7.2. Steady-state kinetics with phospho(enol)pyruvate (PEP) as the variable substrate show that there is a threefold decrease in the Km for PEP in the presence of 1.0 mM fructose-1,6-diphosphate (FDP), and that the activity of PH PyK is increased over fourfold by FDP at low (0.1 mM) PEP concentrations. Lineweaver-Burk plots are linear in the presence and absence of FDP, indicating that the Michaelis-Menten curves are hyperbolic. The amino acid composition for pig heart PyK shows close similarities between pig muscle and kidney PyKs, but not liver PyK. Among the data on pI, pHo, and FDP activation, only the activation by FDP is useful in tentatively designating pig heart PyK as an M2 isozyme.
Kiffmeyer
W R
WR
Department of Anatomy, Indiana University, Bloomington 47405.
Farrar
W W
WW
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine; D014018:Tissue Distribution
D000596:Amino Acids; D007527:Isoenzymes; D011770:Pyruvate Kinase