Purification and properties of hepatic glutamine synthetases from the ureotelic gulf toadfish, Opsanus beta

Abstract

The cytoplasmic and mitochondrial forms of glutamine synthetase (GSase) were purified from the liver of the gulf toadfish Opsanus beta by modifications of methods previously applied to dogfish shark to examine their kinetic and structural properties. Both isozymes have subunit molecular weights of approximately 42 kDa (by SDS-PAGE) and native molecular weights of approximately 365 kDa (by gel filtration chromatography), suggesting an octomeric arrangement of the native enzymes. Identity of the purified proteins as GSase was further confirmed by western blot analysis using rabbit anti-chicken GSase antibodies. The requirement for MgCl 2 and several kinetic properties (e.g.,K ms for glutamate, ATP and ammonia) of the two isozymes were very similar. Also notable was that both isozymes had K ms for ammonia in the micromolar range (like the dogfish enzyme). These results suggest that the enzymes are probably easily saturated with ammonia under physiological conditions. The two GSase isozymes differed substantially in terms of inhibition by methionine sulfoximine, pH optima, specific activity and ratios of transferase to biosynthetic activities. Given the similarities in size, these results suggest that the molecular model of a single gene coding for both isozymes as has been demonstrated in the dogfish shark may not apply to the toadfish GSases.