Mitochondrial malic enzymes. Purification and properties of the NAD(P)-dependent malic enzyme from canine small intestinal mucosa.
An NAD(P)-dependent malic enzyme with a specific activity of 40.6 mumol of NADH/min/mg of protein and an isoelectric point of 5.4 was purified to apparent homogeneity from canine small intestinal mucosal mitochondria. The purification procedure employed ammonium sulfate fractionation, Sepharose CL 6B gel filtration, chromatography on DEAE-cellulose to remove the interfering malate dehydrogenase, and affinity chromatography on 2',5'-ADP-Sepharose and NAD-agarose to take advantage of the dual coenzyme specificity. Antibody prepared from the purified enzyme produced a single peak upon cross-rocket immunoelectrophoresis against the mitochondrial sonicate. Continuous polyacrylamide gel electrophoresis showed NAD and NADP activity co-migrating with the native protein band. A single band of protein having an apparent Mr = 62,000 was seen on sodium dodecyl sulfate electrophoresis. At pH 7.3, gel filtration revealed a single peak of activity with NAD and NADP corresponding to an apparent Mr = 282,000. Gradient gel polyacrylamide electrophoresis at pH 9.0 indicated an additional broad band of activity corresponding to a Mr = 141,000. Under physiological conditions therefore the protein appears to exist as a tetramer of Mr = 282,000 composed of four equal subunits, whereas at elevated pH values during electrophoresis, partial dissociation to a dimeric species occurs.
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007122:Immunoelectrophoresis; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000201:enzymology*; D007525:Isoelectric Focusing; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism*
D009249:NADP; D008291:Malate Dehydrogenase