Drincovich M F, Andreo C S
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), (CONICET, Fund M Lillo, Universidad Nacional de Rosario), Argentina.
Biochem Mol Biol Int. 1995 Aug;36(6):1287-97.
NADP-malic enzyme from maize leaves is covalently labeled with a fluorescent-SH reactive probe eosin-5-maleimide (EMA), which reacts with groups that are totally protected by NADP against inactivation. The comparison of the emission fluorescence spectra of the native and the modified enzyme suggests the proximity of the fluorescent groups of the native enzyme (probably tryptophanyl groups) and the EMA modified residues. Intrinsic fluorescence quenching studies shows that NADP is the only substrate capable to interact with the fluorescent excited groups of the enzyme, while Mg2+ is able to increase this interaction. Quenching studies of EMA-bound fluorescence shows that the NADP-binding site was modified and thus uncapable of further interaction with the nucleotide. When the results of protection studies are combined with those of extrinsic quenching experiments, we must conclude that EMA reacts with sulfhydryl groups that are involved in the NADP-binding site of the enzyme.
来自玉米叶片的NADP-苹果酸酶用荧光-SH反应探针曙红-5-马来酰亚胺（EMA）进行共价标记，EMA与完全受NADP保护而不被灭活的基团发生反应。天然酶和修饰酶的发射荧光光谱比较表明，天然酶的荧光基团（可能是色氨酸基团）与EMA修饰的残基接近。内源荧光猝灭研究表明，NADP是唯一能够与酶的荧光激发基团相互作用的底物，而Mg2+能够增强这种相互作用。EMA结合荧光的猝灭研究表明，NADP结合位点被修饰，因此无法与核苷酸进一步相互作用。当保护研究结果与外源猝灭实验结果相结合时，我们必须得出结论，EMA与参与酶NADP结合位点的巯基发生反应。
