Klinger E, Aviram I
Department of Biochemistry, Faculty of Life Sciences, Tel-Aviv University, Israel.
Biochem J. 1992 Jul 15;285 ( Pt 2)(Pt 2):635-9. doi: 10.1042/bj2850635.
Activation of superoxide-producing NADPH oxidase of neutrophils requires the presence of cell membranes, cytosolic components and arachidonate and is markedly enhanced by non-hydrolysable analogues of guanine nucleotides, i.e. guanosine 5'-[gamma-thio]triphosphate and guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG). Gel filtration and ultrafiltration of the cytosol decreased the basal activity of NADPH oxidase. Activity could be restored by GTP, suggesting participation of the nucleotide in basal activation. Preincubation of neutrophil cytosol with periodate-oxidized p[NH]ppG (ox-p[NH]ppG) followed by gel filtration resulted in a time-dependent enhancement of basal oxidase activity. The presence of GDP or GTP, but not ATP, during the incubation with ox-p[NH]ppG abolished this enhancement. These data are consistent with a stable association of ox-p[NH]ppG with an oxidase-linked cytosolic protein. SDS/PAGE of neutrophil cytosol preincubated with [3H]ox-p[NH]ppG revealed radioactivity in bands migrating as 100, 70, 47, 34 and 22 kDa proteins. Evidence for covalent labelling of the cytosolic protein p47-phox with [3H]ox-p[NH]ppG is presented. Heterogeneity of cytosolic GTP-binding sites and possible participation of protein p47-phox in functional interaction with GTP analogues during cell-free activation are suggested.
中性粒细胞产生超氧化物的NADPH氧化酶的激活需要细胞膜、胞质成分和花生四烯酸的存在，并且鸟嘌呤核苷酸的不可水解类似物，即鸟苷5'-[γ-硫代]三磷酸和鸟苷5'-[βγ-亚氨基]三磷酸（p[NH]ppG）可显著增强其活性。对胞质溶胶进行凝胶过滤和超滤会降低NADPH氧化酶的基础活性。GTP可恢复该活性，表明核苷酸参与基础激活过程。用高碘酸盐氧化的p[NH]ppG（ox-p[NH]ppG）对中性粒细胞胞质溶胶进行预孵育，随后进行凝胶过滤，结果显示基础氧化酶活性呈时间依赖性增强。在与ox-p[NH]ppG孵育期间存在GDP或GTP（而非ATP）可消除这种增强作用。这些数据与ox-p[NH]ppG与氧化酶相关的胞质蛋白稳定结合一致。用[3H]ox-p[NH]ppG预孵育的中性粒细胞胞质溶胶的SDS/PAGE显示，在迁移为100、70、47、34和22 kDa蛋白质的条带中有放射性。本文提供了[3H]ox-p[NH]ppG对胞质蛋白p47-phox进行共价标记的证据。提示了胞质GTP结合位点的异质性以及蛋白p47-phox在无细胞激活过程中与GTP类似物功能相互作用中的可能参与。
