Feltner D E, Smith R H, Marasco W A
J Immunol. 1986 Sep 15;137(6):1961-70.
We have characterized the GTPase activity of the Ni-like guanine-nucleotide-binding regulatory protein in rabbit neutrophil plasma membranes. The low Km (3.64 +/- 0.87 X 10(-7) M) GTPase copurified with the formyl peptide receptor in the plasma membrane fraction obtained by discontinuous sucrose density gradient centrifugation. The Vmax (23.9 +/- 2.91 pmol/mg/min) and Km of the unstimulated enzyme were similar to those reported for Ni in other cell types. The activity of the unstimulated enzyme was both magnesium and sodium dependent and linear over the first 4 min of the assay. The chemoattractants, formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and leukotriene B4 (LTB4) stimulated the GTPase in purified neutrophil plasma membrane preparations, whereas other secretagogues, such as A23187 and PMA, were without effect. Lineweaver-Burk analysis showed an fMLP-induced increase in Vmax (31.94 +/- 4.80 pmol/mg/min) (33.1 +/- 9.5%) but not in Km. The dose-response curve for fMLP stimulation showed an ED50 of 4.1 +/- 1.0 X 10(-8) M and an overall 22.2 +/- 3.1% maximal stimulation. C5a (30 micrograms/ml) increased the activity of the GTPase 21.3 +/- 5.7% and 10(-7) M LTB4 produced a 32.2 +/- 5.4% increase. Activated pertussis toxin treatment of neutrophil plasma membranes inhibited by 72.5 +/- 14.3% the stimulation of GTPase activity induced by fMLP; however, activated cholera toxin had no effect on the inhibition of fMLP stimulation, suggesting a direct role for an Ni-like protein in the coupling process. In contrast to the lack of inhibition of fMLP stimulation by activated cholera toxin treatment of plasma membranes, both pertussis toxin and to a lesser extent cholera toxin treatment reduced fMLP, C5a, and LTB4 stimulation of the GTPase in sonicates prepared from pretreated whole cells. Pertussis toxin inhibited fMLP stimulation of the GTPase by 75 +/- 7%, C5a stimulation was inhibited by 83 +/- 13%, and LTB4 stimulation was inhibited completely. Sonicates prepared from neutrophils treated similarly with cholera toxin showed a smaller inhibition of GTPase activity (50 +/- 4% and 14 +/- 9% for fMLP and LTB4, respectively) with the exception of C5a, where CT inhibition (81 +/- 32%) equaled pertussis toxin inhibition. Similarly, pertussis toxin completely inhibited the release of the granule enzyme N-acetyl-glucosaminidase by all three chemoattractants, whereas cholera toxin, except with C5a stimulation, had little or no effect.(ABSTRACT TRUNCATED AT 400 WORDS)
我们已对兔中性粒细胞质膜中类Ni鸟嘌呤核苷酸结合调节蛋白的GTP酶活性进行了表征。通过不连续蔗糖密度梯度离心获得的质膜组分中，低Km（3.64±0.87×10⁻⁷M）的GTP酶与甲酰肽受体共同纯化。未刺激酶的Vmax（23.9±2.91 pmol/mg/min）和Km与其他细胞类型中报道的Ni相似。未刺激酶的活性既依赖镁也依赖钠，且在测定的前4分钟呈线性。趋化因子甲酰甲硫氨酰亮氨酰苯丙氨酸（fMLP）、C5a和白三烯B4（LTB4）刺激纯化的中性粒细胞质膜制剂中的GTP酶，而其他促分泌剂，如A23187和佛波酯（PMA）则无作用。Lineweaver - Burk分析显示，fMLP诱导Vmax增加（31.94±4.80 pmol/mg/min）（33.1±9.5%），但Km未增加。fMLP刺激的剂量反应曲线显示ED50为4.1±1.0×10⁻⁸M，最大刺激总体为22.2±3.1%。C5a（30微克/毫升）使GTP酶活性增加21.3±5.7%，10⁻⁷M的LTB4使活性增加32.2±5.4%。用活化百日咳毒素处理中性粒细胞质膜可抑制fMLP诱导的GTP酶活性刺激的72.5±14.3%；然而，活化霍乱毒素对fMLP刺激的抑制无作用，这表明类Ni蛋白在偶联过程中起直接作用。与活化霍乱毒素处理质膜对fMLP刺激缺乏抑制相反，百日咳毒素以及程度较轻的霍乱毒素处理均降低了预处理全细胞制备的超声裂解物中fMLP、C5a和LTB4对GTP酶的刺激。百日咳毒素抑制fMLP对GTP酶的刺激达75±7%，C5a刺激被抑制83±13%，LTB4刺激被完全抑制。用霍乱毒素类似处理中性粒细胞制备的超声裂解物显示对GTP酶活性的抑制较小（fMLP和LTB4分别为50±4%和14±9%），C5a除外，其霍乱毒素抑制（81±32%）与百日咳毒素抑制相当。同样，百日咳毒素完全抑制所有三种趋化因子诱导的颗粒酶N - 乙酰葡糖胺酶的释放，而霍乱毒素除对C5a刺激外，几乎没有或没有作用。（摘要截断于400字）
