| Abstrakt: |
Photoaffinity labeling with [α-32P]-8-azidoadenosine 5-diphosphate (8N3ADP) and [β-32P]-2-azidoadenosine 5-diphosphate (2N3ADP) was used to identify overlapping tryptic and chymotryptic generated peptides within the adenine binding domain of the regulatory ADP site of bovine liver glutamate dehydrogenase (GDH). In the absence of UV irradiation, 8N3ADP was able to activate the reverse reaction catalyzed by GDH as well as ADP. Photoinsertion of both [α32P]8N3ADP and [β32P]2N3ADP was reduced best by ADP in comparison to other nucleotides. Photolabeling of GDH with [α32P]8N3ADP appeared to be biphasic, with saturation occurring near 80 and 130 μM, whereas [β32P]2N3ADP showed saturation near 50 μM. When 60 μM [α32P]8N3ADP (below the first saturation value) was used to identify peptides within the ADP binding domain, peptides corresponding to residues G156−K200 and E175−K200 (tryptic) and I158−Y183 (chymotryptic) were photolabeled. However, when 160 μM [α32P]8N3ADP (above the second saturation value) was used, the peptide D403−R418 was also photolabeled. Digestion with both trypsin and chymotrypsin resulted in isolation of peptides E175−Y183 and A184−I192. [β32P]2N3ADP at 90 μM also photolabeled tryptic peptides G156−K200 and C270−K289. C270−K289 was shown earlier to be within the NAD+ binding site [Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2, 142−147]. These results are consistent with the residues E175−I192 being within the adenine binding domain of the ADP regulatory site. |
