Association-dissociation studies of bovine and rat liver glutamic dehydrogenase by high-performance liquid chromatography gel filtration

Abstract

The concentration-dependent association-dissociation tendency of purified bovine liver and rat liver glutamic dehydrogenase (GDH) has been demonstrated by high-performance liquid Chromatographic gel filtration. In the concentration range of 100 to 1.0 μg bovine GDH/ml molecular species ranged from dimer and unimer to subunimeric forms. The dissociation process of the unimeric hexapeptide, consisting of six polypeptide chains, to the subunimeric tripeptide, consisting of three polypeptide chains, was irreversible without added ionic support, but reversible with added ionic support. In dilute Tris-HCl bovine liver GDH was dispersed to subunimeric sizes. Increasing the ionic strength in 20 m m phosphate as the mobile phase increased dissociation to a subunimeric tripeptide while sustaining as much as 80% of its activity. Activity of a eluting subunimer was verified by the inclusion of reaction substrates (NAD and glutamute) in the mobile phase and quantification of reaction products (NADH) in chromatograms. Gel filtration of GDH in the presence of GTP with NADH rendered a subunimeric tripeptide, largely independent of ionic strength or GDH concentration. Rat liver GDH, differing from bovine liver GDH, was dissociated by gel filtration to an active tripeptide independent of ionic or buffer conditions.