Shoemaker M T, Haley B E
Department of Biochemistry, College of Medicine, College of Pharmacy, University of Kentucky Medical Center, Lexington.
Bioconjug Chem. 1996 May-Jun;7(3):302-10. doi: 10.1021/bc960014n.
Photoaffinity labeling with [alpha-32P]-8-azidoadenosine 5'-diphosphate (8N3ADP) and [beta-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 [alpha 32P] 8N3ADP and [beta 32P]2N3ADP was reduced best by ADP in comparison to other nucleotides. Photolabeling of GDH with [alpha 32P]8N3-ADP appeared to be biphasic, with saturation occurring near 80 and 130 microM, whereas [beta 32P]2N3ADP showed saturation near 50 microM. When 60 microM [alpha 32P]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 microM [alpha 32P]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. [beta 32P]2N3ADP at 90 microM 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-[192 being within the adenine binding domain of the ADP regulatory site.
利用[α-32P]-8-叠氮腺苷5'-二磷酸（8N3ADP）和[β-32P]-2-叠氮腺苷5'-二磷酸（2N3ADP）进行光亲和标记，以鉴定牛肝谷氨酸脱氢酶（GDH）调节性ADP位点的腺嘌呤结合域内胰蛋白酶和糜蛋白酶产生的重叠肽段。在没有紫外线照射的情况下，8N3ADP能够激活GDH催化的逆反应以及ADP。与其他核苷酸相比，ADP对[α-32P] 8N3ADP和[β-32P]2N3ADP的光插入作用抑制效果最佳。用[α-32P]8N3-ADP对GDH进行光标记似乎呈双相性，在80和130微摩尔附近出现饱和，而[β-32P]2N3ADP在50微摩尔附近显示饱和。当使用60微摩尔[α-32P]8N3ADP（低于第一个饱和值）来鉴定ADP结合域内的肽段时，对应于残基G156-K200和E175-K200（胰蛋白酶）以及I158-Y183（糜蛋白酶）的肽段被光标记。然而，当使用160微摩尔[α-32P]8N3ADP（高于第二个饱和值）时，肽段D403-R418也被光标记。用胰蛋白酶和糜蛋白酶消化都导致分离出肽段E175-Y183和A184-I192。90微摩尔的[β-32P]2N3ADP也对胰蛋白酶肽段G156-K200和C270-K289进行了光标记。先前已表明C270-K289在NAD+结合位点内[Kim, H., and Haley, B. (1991) Bioconjugate Chem. 2, 142-147]。这些结果与残基E175-[192在ADP调节位点的腺嘌呤结合域内一致。
