Isolation and partial purification of dicarboxylic acid binding protein from luminal-membrane vesicles of rabbit...

A specific dicarboxylic acid binding protein was isolated by solubilizing highly purified renal luminal-membrane vesicles with the non-ionic detergent C12E8 , followed by affinity chromatographic procedures. SDS-polyacrylamide gel electrophoresis of the samples containing dicarboxylic acid binding protein showed a single sharp band of an apparent molecular weight of 50 000. After treatment with mercaptoethanol the protein was split in two subunits of apparent molecular weights of 35 000 and 15 000. By analytical ultracentrifugation the minimal molecular weight of the dicarboxylic acid binding protein preparation was calculated to be 54 000. Binding of the radioactive succinate and L-malate to the dicarboxylic acid binding protein preparation as studied by equilibrium dialysis showed saturation phenomenon and was specifically inhibited by addition of D-malate. The dissociation constants for succinate (0.18 mM) and L-malate (0.33 mM) calculated from the binding data agree extremely well with the apparent Km values for these organic acids found in transport studies utilizing intact luminal-membrane vesicles.