Correlation between activation and dimer formation of rat renal phosphate-dependent glutaminase.
Gel filtration and velocity sedimentation in sucrose gradients were used to determine the molecular weights of purified rat renal phosphate-dependent glutaminase. The purified glutaminase has a molecular weight of 160,000 in Tris or barbital buffers and forms dimers of 332,000 molecular weight in the presence of its activator, Pi. The correlation between activation and dimer formation was investigated by determining the sedimentation coefficient at various concentrations of glutaminase activators. Saturation curves for Pi and riboflavin phosphate demonstrate an excellent correlation between per cent activation and increasing S20,w with increasing concentrations of these activators. The concentrations required for half-maximal saturation were 40 to 50 mM for Pi and 10 to 15 mM for riboflavin phosphate. Correlation between activation and dimer formation was also found with other activators at subsaturating concentrations. Moreover, the activation and dimer formation were found to be reversed to a similar extent by increasing concentrations of NaCl. Finally, we studied the effects of Pi and NaCl on the stability of glutaminase activity at 37 degrees. Pi stabilized glutaminase activity by increasing the t1/2 for inactivation from 12 min in the absence of Pi to 242 at 150 mM Pi. The concentration of Pi which gave approximately half-maximal change in t1/2 was 50 mM and addition of NaCl reversed this stabilization. These results support the hypothesis that phosphate-dependent glutaminase is active only as a dimer or larger aggregate. However, we cannot exclude the possibility that binding of Pi changes the monomer conformation sufficiently to produce activation and that this new conformation leads to self-association.
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005972:Glutaminase / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010710:Phosphates / Q000494:pharmacology*; D051381:Rats
D046911:Macromolecular Substances; D010710:Phosphates; D005972:Glutaminase