Mouse liver cytoplasmic malate dehydrogenase: Rapid isolation, characterization and immunochemical comparison with hepatoma enzyme

Abstract

Cytoplasmic NAD-dependent malate dehydrogenase is decreased in activity in three transplantable mouse hepatomas compared to the activity of this enzyme in liver tissue. This enzyme is composed of several molecular forms of similar size which differ slightly in charge; the total activity and the discernible number of forms of the enzyme are decreased in both hepatoma and fetal liver. Mixing experiments suggest the absence of a significant quantity of unbound inhibitor of enzyme activity in the tumor or an activator in the liver. The liver cytoplasmic enzyme was purified to homogeneity by a relatively rapid method using Blue Sepharose affinity chromatography, which results in a good yield and high specific activity of the enzyme. Cytoplasmic and mitochondrial enzymes bind and elute differentially from this affinity resin. Molecular weight, kinetic constants and amino acid composition of the cytoplasmic enzyme were determined. Monospecific antiserum to the cytoplasmic enzyme has been produced in a goat and used to demonstrate a lack of immunological cross-reactivity between the mitochondrial and cytoplasmic enzyme. The tumor and liver cytoplasmic enzymes possess similar, if not identical, immunological determinants. Immunotitration experiments have been used to demonstrate that liver and hepatoma enzyme are identical in specific activity. Thus, the reduction in level of cytoplasmic enzyme in hepatoma is due to a decrease in the number of molecules per tissue mass.