Binding of ATP to brain glutamate decarboxylase as studied by affinity chromatography.

The interactions of two forms of porcine brain glutamate decarboxylase (beta-GAD and gamma-GAD) with the effector ATP were studied by affinity chromatography. A third form, alpha-GAD, was only slightly retarded by the affinity matrix and was eluted in the buffer wash. The interaction of GAD with the ATP affinity matrix was qualitatively similar to its interaction with free ATP as reported in previous kinetic studies. The rank order of adenine nucleotides as eluting agents and affinity ligands was ATP greater than ADP greater than AMP. GAD was also eluted by its cofactor, pyridoxal 5'-phosphate, and this was enhanced by 1 mM Pi. In contrast, a high concentration (140 mM) of Pi by itself was required to elute the enzyme. GAD remained active while bound to the affinity column and was eluted in the holoenzyme form by ATP, indicating that the affinity ligand did not bind in the active site and did not displace catalytically active cofactor from the enzyme.