Rapid purification and radioimmunoassay of cytosolic malic enzyme.
A very rapid and highly effective procedure has been devised for the isolation of homogeneous malic enzyme from rat liver cytosol. A combination of precipitation with 10 to 20% polyethylene glycol, ion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Procion Red HE-3B Agarose was used to prepare 3 to 4 mg of homogeneous malic enzyme from the livers of two rats in 18 h. In addition to introducing the advantages of simplicity, speed, and high yield (31%) the new method eliminates potentially denaturing steps (heat treatment, ethanol fractionation) and prolonged dialysis procedures used in other purification schemes. Malic enzyme purified by this new method was use to immunize rabbits. The resulting antibodies bound purified rat liver and mouse liver malic enzymes with very similar affinities and also avidly complexed cytosolic malic enzyme from two murine cell lines, 3T3-L1 preadipocytes and 3T3-C2 fibroblasts. When purified malic enzyme was incubated with lactoperoxidase, glucose oxidase and Na 125I 1.8 atoms of 125I were incorporated per molecule of enzyme with full retention of catalytic activity, subunit size, and immunoreactivity. The antiserum, the purified enzyme, and enzymatically iodinated 125I-malic enzyme were used to construct a sensitive, competitive binding radioimmunoassay for the measurement of malic enzyme mass in the range of 1 to 100 ng.
D000818:Animals; D001667:Binding, Competitive; D011232:Chemical Precipitation; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D008099:Liver / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D051379:Mice; D008832:Microchemistry; D011485:Protein Binding; D011863:Radioimmunoassay; D051381:Rats
D008291:Malate Dehydrogenase