IDENTIFICATION OF A PEPTIDE OF THE GUANOSINE TRIPHOSPHATE-BINDING SITE WITHIN BRAIN GLUTAMATE-DEHYDROGENASE ISOPROTEINS USING 8-AZIDOGUANOSINE TRIPHOSPHATE

Photoaffinity labeling with [gamma-P-32]8N(3)GTP (8-azidoguanosine tri phosphate) was used to identify the guanine binding peptides of the GT P binding site within two types of glutamate dehydrogenase isoproteins (GDH I and GDH II) isolated from bovine brain. 8N(3)GTP, without phot olysis, mimicked the inhibitory properties of GTP on GDH I and GDH II activities, Saturation of photoinsertion of GDH isoproteins revealed a n apparent K-d of 8 mu M (GDH I) and 24 mu M (GDH II) for [gamma-P-32] 8N(3)GTP. Ion-exchange and reversed-phase high-performance liquid chro matography (HPLC) were used to isolate photolabel-containing peptides generated with trypsin. This identified a portion of the guanine bindi ng domain within the GTP binding site as the region containing the seq uence I-S-G-A-S-E-X-D-I-V-H-S-A-L-A-Y-T-M-E-R (GDH I) and I-S-G-A-S-E- X-D-I-V-H-S-G-L-A-Y-T-M-E-R (GDH II). The symbol X indicates a positio n for which no phenylthiohydantoin-amino acid could be assigned. The m issing residue, however, can be designated as a photolabeled lysine si nce the sequences including the lysine residue in question have a comp lete identity with those of the other GDH species known. Also, trypsin was unable to cleave the photolabeled peptide at this site, Photolabe ling of these peptides was prevented by the presence of GTP during pho tolysis, while other nucleotides could not reduce the amount of photoi nsertion as effectively as GTP. These results demonstrate selectivity of the photoprobe for the GTP binding site and suggest that the peptid e identified using the photoprobe is located in the GTP binding domain of the brain GDH isoproteins.