Hirst B H, Forte J G
Biochem J. 1985 Nov 1;231(3):641-9. doi: 10.1042/bj2310641.
When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+)-ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation.
从静息壁细胞中分离时，大部分（H⁺+K⁺）-ATP酶活性在微粒体部分中得以恢复。这些微粒体囊泡显示出低K⁺通透性，因此加入缬氨霉素会显著刺激（H⁺+K⁺）-ATP酶活性以及质子积累。从受刺激的壁细胞中分离时，（H⁺+K⁺）-ATP酶重新分布到更大、更致密的囊泡中：即刺激相关（s.a.）囊泡。S.a.囊泡显示出K⁺通透性增加，因此在低K⁺浓度下观察到最大的（H⁺+K⁺）-ATP酶和质子积累活性，并且加入缬氨霉素后活性没有增强。（H⁺+K⁺）-ATP酶亚细胞分布的变化与壁细胞受刺激时观察到的形态变化相关，微粒体和s.a.囊泡分别源自细胞内小管泡和顶端质膜。从受刺激的胃黏膜中可回收的总（H⁺+K⁺）-ATP酶活性是静息组织的64%。因此，我们测试了s.a.囊泡中的潜在活性。用辛基葡糖苷使s.a.囊泡通透化后，（H⁺+K⁺）-ATP酶活性增加了2倍以上。潜在的（H⁺+K⁺）-ATP酶活性对高度胰蛋白酶处理有抗性（这种处理使胃微粒体中的所有活性失活）。约20%的非潜在（H⁺+K⁺）-ATP酶活性也对胰蛋白酶消化有抗性。我们将这些结果解释为表明，在s.a.囊泡中，约55%具有外翻取向且对ATP不通透，10%外翻且对ATP通透，35%具有内翻取向。
