title: Homogeneous pyruvate kinase isolated from yeast by two different methods is indistinguishable from pyruvate kinase in cell-free extract.
abstract: In this report, we have compared homogeneous yeast (Saccharomyces cerevisiae) pyruvate kinase to enzyme from cell-free extracts in several different ways: 1) isoelectric focusing of cell-free extracts indicates one peak of pyruvate kinase activity whose isoelectric point is the same as that of the pure enzyme; 2) antibody prepared to the pure enzyme produces a single, fused precipitin line against enzyme in the cell-free extract and pure enzyme; 3) immunoelectrophoresis of cell-free extract produces one precipitin arc which has the same mobility as that of the pure enzyme; and 4) immunoprecipitation of the pure enzyme from cell-free extract with subsequent solubilization in 1% sodium dodecyl sulfate and electrophoresis on sodium dodecyl sulfate-polyacrylamide gels produces a single protein band attributable to pyruvate kinase which co-migrates with the purified enzyme. Within the limits of the sensitivity of the methods employed, we conclude that the homogeneous pyruvate kinase prepared from yeast lysed either by Manton-Gaulin homogenization (Aust, A., Yun, S.-L., and Suelter, C. (1975) Methods Enzymol. 42, 176-182) or by toluolysis (Yun, S.-L., Aust, A.E., and Suelter, C.H. (1977) J. Biol. Chem. 251, 124-128) is identical with pyruvate kinase in cell-free extract.
mesh_terms: D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007700:Kinetics; D008970:Molecular Weight; D011233:Precipitin Tests; D011770:Pyruvate Kinase / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D011770:Pyruvate Kinase