Cheng Ming, Guillory Richard John
Department of Biochemistry and Biophysics, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu 96822, USA.
J Biochem Mol Biol Biophys. 2002 Jun;6(3):177-84. doi: 10.1080/1025814021000000925.
Functional reagents known to bring about the formation of a distinct membrane molecular complex of the subunits of cytochrome b(558) (gp 91(phox) and p22(phox)) were investigated for their influence on the O2- generating capability of liposome incorporated cytochrome b(558) preparations. One, ethyleneglycolbis[sulfo-succinimidylsuccinate], (sulfo-EGS) was found to inhibit O2- generation at concentrations which are known to result in cross-linking the two subunits of cytochrome b(558). Sulfosuccinimidyl [4-azidophenyldithio] propionate, (sulfo-SADP) on the other hand, was found to be a powerful inhibitor of the cytochrome b(558) dependent O2- production at concentrations not able to result in cross linking of the two subunits. Sulfo-SADP inhibits the cytochrome b(558) O2- production 50% at 25 microM, while sulfo-EGS requires 400 microM. For these reagents, the succinimidyl group of sulfo-SADP and sulfo-EGS is the reactive group, which inhibit irreversibly, cytochrome b(558) generation of O2-. Both sulfo-SADP and sulfo-EGS have similar linker arms of 13.9 and 16.1 A, respectively. The difference, accounting for the strong inhibitory profile for sulfo-SADP as compared with sulfo-EGS, resides in the aryl group associated with the sulfo-SADP. The aryl group of sulfo-SADP has been found to be important in directing the specificity of the probe in its inhibition of O2- generation. When the disulfide bond linking the aromatic portion of the probe to the succinimidyl ring is cleaved by DTT (dithiothreitol), the product loses its specificity and has an inhibitory activity with respect to O2- generation comparable to that of sulfo-EGS. The partial protection against the inhibitory influence of sulfo-SADP by NADP(+) indicates that the reagent may interact at the pyridine nucleotide-binding domain of cytochrome b(558). Its low inhibitory titer and its water solubility suggest that sulfo-SADP reacts with a specific amine (the primary reactant for the succinimidyl group) on cytochrome b(558).
研究了已知能促使细胞色素b(558)（gp91(phox)和p22(phox)）亚基形成独特膜分子复合物的功能试剂对脂质体包裹的细胞色素b(558)制剂产生超氧阴离子（O2-）能力的影响。其中一种试剂，乙二醇双[磺基琥珀酰亚胺琥珀酸酯]（sulfo-EGS），在已知会导致细胞色素b(558)两个亚基交联的浓度下，能抑制超氧阴离子的产生。另一方面，磺基琥珀酰亚胺[4-叠氮苯基二硫代]丙酸酯（sulfo-SADP）在无法导致两个亚基交联的浓度下，被发现是细胞色素b(558)依赖性超氧阴离子产生的强力抑制剂。sulfo-SADP在25微摩尔浓度时能抑制细胞色素b(558)超氧阴离子产生50%，而sulfo-EGS则需要400微摩尔。对于这些试剂，sulfo-SADP和sulfo-EGS的琥珀酰亚胺基团是反应性基团，它们不可逆地抑制细胞色素b(558)产生超氧阴离子。sulfo-SADP和sulfo-EGS的连接臂分别相似，长度为13.9埃和16.1埃。与sulfo-EGS相比，sulfo-SADP具有更强抑制作用的差异在于与sulfo-SADP相关的芳基。已发现sulfo-SADP的芳基在引导探针抑制超氧阴离子产生的特异性方面很重要。当连接探针芳香部分与琥珀酰亚胺环的二硫键被二硫苏糖醇（DTT）裂解时，产物失去其特异性，并且对超氧阴离子产生的抑制活性与sulfo-EGS相当。烟酰胺腺嘌呤二核苷酸磷酸（NADP(+)）对sulfo-SADP抑制作用的部分保护表明，该试剂可能在细胞色素b(558)的吡啶核苷酸结合结构域相互作用。其低抑制效价和水溶性表明，sulfo-SADP与细胞色素b(558)上的特定胺（琥珀酰亚胺基团的主要反应物）发生反应。
