Pick E, Gorzalczany Y, Engel S
Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel.
Eur J Biochem. 1993 Oct 1;217(1):441-55. doi: 10.1111/j.1432-1033.1993.tb18264.x.
Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes requires the interaction of membrane-associated cytochrome b559 with three cytosolic components; p47-phox, p67-phox and sigma 1. We proposed that sigma 1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP-binding protein (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of sigma 1 and demonstrate that the NADPH-oxidase-activating capacity is associated, throughout the purification sequence, with a protein binding 35S-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma 1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22-kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and anti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the sigma 1 heterodimer could be separated by reverse-phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22-kDa protein with rac1 p21 and that of the 24-kDa protein with rho GDI. Purified heterodimeric sigma 1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma 1 heterodimer with 1% sodium cholate, followed by gel filtration or anion-exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell-free O2- generation. Artificial heterodimeric sigma 1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native sigma 1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ solution. Under similar conditions, heterodimeric sigma 1 was resistant to nucleotide exchange.(ABSTRACT TRUNCATED AT 400 WORDS)
吞噬细胞中产生超氧化物（O₂⁻）的NADPH氧化酶的激活需要膜相关细胞色素b559与三种胞质成分相互作用；即p47⁻phox、p67⁻phox和sigma 1。我们曾提出sigma 1是由22 kDa和24 kDa蛋白质组成的异二聚体，它们初步被鉴定为小GTP结合蛋白（G蛋白）rac1 p21和rho的GDP解离抑制剂（rho GDI）。我们现在描述一种改良的快速纯化sigma 1的方法，并证明在整个纯化过程中，NADPH氧化酶激活能力与一种结合³⁵S标记的鸟苷5'-[3-O-硫代]三磷酸的蛋白质相关。SDS/PAGE分析证实sigma 1活性与22 kDa和24 kDa蛋白质的存在绝对相关。用一系列针对小G蛋白的抗体进行免疫印迹表明，22 kDa蛋白质仅被与rac1 p21反应的抗体识别；未发现与抗（rac2 p21）、抗[v-ras(H) p21]和抗（rap1 p21）的反应。在胞质分级分离的任何阶段都未检测到游离的rac1 p21（不与rho GDI形成复合物）。组成sigma 1异二聚体的蛋白质可以通过反相色谱分离，并对每种分离蛋白质经胰蛋白酶消化产生的肽段进行氨基酸测序。这证明了22 kDa蛋白质与rac1 p21相同，24 kDa蛋白质与rho GDI相同。在确保无游离核苷酸的条件下，纯化的异二聚体sigma 1的活性不需要外源GTP。用1%胆酸钠处理sigma 1异二聚体，然后在1%胆酸钠存在下进行凝胶过滤或阴离子交换色谱，可有效地将rac1 p21与rho GDI分离。通过这些方法获得的单体rac1 p21能够刺激无细胞体系中O₂⁻的产生。能够激活NADPH氧化酶的人工异二聚体sigma 1可以通过将纯化的单体rac1 p21和rho GDI重组并去除用于解离天然sigma 1二聚体的胆酸钠在体外重建。在低Mg²⁺溶液中去除内源性核苷酸后，单体rac1 p21对外源GTP表现出几乎绝对的依赖性。在类似条件下，异二聚体sigma 1对核苷酸交换具有抗性。（摘要截短至400字）
