Photoaffinity labeling of brain glutamate dehydrogenase isoproteins with an azido-ADP

The ADP binding site within two types of bovine brain glutamate dehydrogena se isoproteins (GDH I and GDH II) was identified using photoaffinity labeli ng with [alpha-P-32]8-azidoadenosine 5'-diphosphate (8N(3)ADP), 8N(3)ADP, w ithout photolysis, mimicked the activatory properties of ADP on GDH I and G DH II activities, although maximal activity with 8N(3)ADP was about 75% of maximal ADP-stimulated activity. Saturation of photoinsertion with [alpha-P -32]8N(3)ADP occurred at around 40 similar to 50 mu M photoprobe with appar ent K-d values near 25 and 40 mu M for GDH I and GDH Il, respectively. Phot oinsertion of [alpha-P-32]8N(3)ADP was decreased best by ADP in comparison with other nucleotides. With the combination of immobilized aluminum affini ty chromatography and reversed-phase high performance liquid chromatography , photolabel-containing peptides generated by tryptic digestion were isolat ed. This identified a portion of the adenine ring binding domain of GDH iso proteins as in the region containing the sequence, EMSWIADT-YASTIGHYDIN. Ph otolabeling of the peptide was prevented over 90% by the presence of 1 mM A DP during photolysis, while other nucleotides could not reduce the amount o f photoinsertion as effectively as ADP. These results demonstrate selectivi ty of the photoprobe for the ADP binding site and suggest that the photolab eled peptide with the residues Glu(179)-Asn(197) is within the ADP binding domain of the brain GDH isoproteins.