Klinger E, Sharabani M, Aviram I
Department of Biochemistry, Faculty of Life Sciences, Tel-Aviv University, Israel.
Biochem J. 1993 Oct 15;295 ( Pt 2)(Pt 2):565-70. doi: 10.1042/bj2950565.
The superoxide-generating NADPH oxidase of neutrophils can be activated in a cell-free system consisting of cell membranes, cytosol and an activating detergent (e.g. arachidonate or SDS). It has previously been reported [Aviram and Sharabani (1989) Biochem. Biophys. Res. Commun. 161, 712-719] that a mixture of phosphoinositides (PPIs), as well as the individual inositol lipids, interfere with the activation process. In the present study it is shown that exposure of the cytosol to PPI results in a progressive (t1/2 = 30 s) loss of its oxidase-supporting activity and that Mg2+ ions eliminate this inactivation. Neomycin, previously described as an inhibitor of cell-free activation, counteracted the effect of PPI and vice versa. Fractionation experiments implicated the p67-phox cytosolic component of the oxidase in the association with PPI. PPI blocked activity of recombinant p67-phox also and quenched the fluorescence intensity of its tryptophan residues. It is suggested that PPIs may mediate the interaction of the oxidase with the cytoskeleton and/or with the membrane.
中性粒细胞产生超氧化物的NADPH氧化酶可在由细胞膜、胞质溶胶和一种激活去污剂（如花生四烯酸盐或十二烷基硫酸钠）组成的无细胞体系中被激活。此前已有报道[阿维拉姆和沙拉巴尼（1989年），《生物化学与生物物理学研究通讯》161卷，712 - 719页]，磷酸肌醇（PPIs）混合物以及单个肌醇脂质会干扰激活过程。在本研究中表明，将胞质溶胶暴露于PPI会导致其氧化酶支持活性逐渐丧失（t1/2 = 30秒），并且镁离子可消除这种失活作用。新霉素，先前被描述为无细胞激活的抑制剂，可抵消PPI的作用，反之亦然。分级分离实验表明氧化酶的p67 - phox胞质成分与PPI相关。PPI也会阻断重组p67 - phox的活性并淬灭其色氨酸残基的荧光强度。有人提出PPIs可能介导氧化酶与细胞骨架和/或与膜的相互作用。
