Gurich R W, Codina J, DuBose T D
Department of Internal Medicine, University of Texas Medical Branch, Galveston 77062.
J Clin Invest. 1991 May;87(5):1547-52. doi: 10.1172/JCI115167.
The effects of guanosine 5'-triphosphate (GTP) and GTP-gamma-S, known activators of GTP binding proteins, on proton transport were investigated in endosome-enriched vesicles (endosomes). Endosomes were prepared from rabbit renal cortex following the intravenous injection of FITC-dextran. The rate of intravesicular acidification was determined by measuring changes in fluorescence of FITC-dextran. Both GTP and GTP-gamma-S stimulated significantly the initial rate of proton transport. In contrast, GDP-beta-S, which does not activate GTP binding proteins, inhibited proton transport. The rank order of stimulation was GTP-gamma-S greater than GTP greater than control greater than GDP-beta-S. GTP-gamma-S stimulation of proton transport was also observed under conditions in which chloride entry was eliminated, i.e., 0 mM external chloride concentration in the presence of potassium/valinomycin voltage clamping. GTP-gamma-S did not affect proton leak in endosomes as determined by collapse of H+ ATPase-generated pH gradients. ADP ribosylation by treatment of endosomal membranes with pertussis toxin revealed two substrates corresponding to the 39-41 kD region and comigrating with alpha i subunits. Pretreatment of the membranes with pertussis toxin had no effect on proton transport in the absence of GTP or GTP-gamma-S. However, pretreatment with pertussis toxin blocked the stimulation of proton transport by GTP. In contrast, as reported in other membranes by others previously, pertussis toxin did not prevent the stimulation of proton transport by GTP-gamma-S. These findings, taken together, indicate that GTP binding proteins are present in endosomal membranes derived from renal cortex and that activation of G protein by GTP and GTP-gamma-S stimulates proton transport in a rank order identical to that reported for other transport pathways modulated by Gi proteins. Therefore, these studies suggest that G proteins are capable of stimulating the vacuolar H ATPase of endosomes directly.
研究了鸟苷 5'-三磷酸（GTP）和 GTP-γ-S（已知的 GTP 结合蛋白激活剂）对富含内体的囊泡（内体）中质子转运的影响。通过静脉注射异硫氰酸荧光素标记的葡聚糖后，从兔肾皮质制备内体。通过测量异硫氰酸荧光素标记的葡聚糖的荧光变化来确定囊泡内酸化速率。GTP 和 GTP-γ-S 均显著刺激质子转运的初始速率。相比之下，不激活 GTP 结合蛋白的 GDP-β-S 则抑制质子转运。刺激的强度顺序为 GTP-γ-S＞GTP＞对照＞GDP-β-S。在消除氯离子内流的条件下，即在存在钾/缬氨霉素电压钳制且外部氯离子浓度为 0 mM 的情况下，也观察到了 GTP-γ-S 对质子转运的刺激作用。如通过 H⁺ATP 酶产生的 pH 梯度的崩溃所确定的，GTP-γ-S 不影响内体中的质子泄漏。用百日咳毒素处理内体膜进行 ADP 核糖基化显示出两种与 39 - 41 kD 区域相对应且与αi 亚基共迁移的底物。在不存在 GTP 或 GTP-γ-S 的情况下，用百日咳毒素预处理膜对质子转运没有影响。然而，用百日咳毒素预处理会阻断 GTP 对质子转运的刺激作用。相比之下，正如其他人先前在其他膜中所报道的，百日咳毒素并不能阻止 GTP-γ-S 对质子转运的刺激作用。综上所述，这些发现表明 GTP 结合蛋白存在于源自肾皮质的内体膜中，并且 GTP 和 GTP-γ-S 对 G 蛋白的激活以与 Gi 蛋白调节的其他转运途径所报道的相同强度顺序刺激质子转运。因此，这些研究表明 G 蛋白能够直接刺激内体的液泡 H⁺ATP 酶。
