Concomitant purification and characterization of malate dehydrogenase, aspartate transaminase, nucleoside diphosphate kinase and enolase from rabbit liver cytosol.

A procedure was developed for purifying the cytosolic isoforms of malate dehydrogenase, aspartate transaminase, enolase and nucleoside diphosphate kinase from a single preparation of rabbit liver. The procedure includes chromatography on reactive-dye, radial-flow columns, and elution of enzymes from columns by substrates, to obtain high yields in a minimal amount of time. The scheme avoids steps used in previous methods that are either difficult to execute in large-scale preparations, or alter the native forms of the enzymes. Examination of the purified enzymes by SDS-PAGE indicated that nearly homogeneous preparations had been obtained. The native molecular weight, subunit molecular weight, and isoelectric point(s) were determined for each enzyme. Although preparations of nucleoside diphosphate kinase purified from cytosol showed only a single band on SDS-PAGE, isoelectric focusing revealed the presence of multiple isoforms.