镁三磷酸腺苷驱动氢离子摄取进入大鼠胰腺腺泡细胞微粒体囊泡组分的特性研究

Characterization of MgATP-driven H+ uptake into a microsomal vesicle fraction from rat pancreatic acinar cells.

作者信息

Thévenod F, Kemmer T P, Christian A L, Schulz I

机构信息

Max-Planck-Institut für Biophysik, Frankfurt/Main, Federal Republic of Germany.

出版信息

J Membr Biol. 1989 Mar;107(3):263-75. doi: 10.1007/BF01871941.

DOI:10.1007/BF01871941

PMID:2469802

Abstract

In microsomal vesicles, as isolated from exocrine pancreas cells, MgATP-driven H+ transport was evaluated by measuring H+-dependent accumulation of acridine orange (AO). Active H+ uptake showed an absolute requirement for ATP with simple Michaelis-Menten kinetics (Km for ATP 0.43 mmol/liter) with a Hill coefficient of 0.99. H+ transport was maximal at an external pH of 6.7, generating an intravesicular pH of 4.8. MgATP-dependent H+ accumulation was abolished by protonophores, such as nigericin (10(-6) mol/liter) or CCCP (10(-5) mol/liter), and by inhibitors of nonmitochondrial H+ ATPases, such as NEM or NBD-Cl, at a concentration of 10(-5) mol/liter. Inhibitors of both mitochondrial and nonmitochondrial H+ pumps, such as DCCD (10(-5) mol/liter) or Dio 9 (0.25 mg/ml), reduced microsomal H+ transport by about 90%. Vanadate (2 x 10(-3) mol/liter), a blocker of those ATPases, which form a phosphorylated intermediate, did not inhibit H+ transport. The stilbene derivative DIDS (10(-4) mol/liter), which inhibits anion transport systems, abolished H+ transport completely. MgATP-dependent H+ transport was found to be anion dependent in the sequence Cl- greater than Br- greater than gluconate-; in the presence of SO2-4, CH3COO- or No-3, no H+ transport was observed. MgATP-dependent H+ accumulation was also cation dependent in the sequence K+ greater than Li+ greater than Na+ = choline+. As shown by dissipation experiments in the presence of different ion gradients and ionophores, both a Cl- and a K+ conductance, as well as a small H+ conductance, were found in the microsomal membranes. When membranes containing the H+ pump were further purified by Percoll gradient centrifugation (ninefold enrichment compared to homogenate), no correlation with markers for endoplasmic reticulum, mitochondria, plasma membranes, zymogen granules or Golgi membranes was found. The present data indicate that the H+ pump located in microsomes from rat exocrine pancreas is a vacuolar- or "V" -type H+ ATPase and has most similarities to that described in endoplasmic reticulum, Golgi apparatus or endosomes.

摘要

从外分泌胰腺细胞分离得到的微粒体囊泡中，通过测量吖啶橙（AO）的H⁺依赖性积累来评估MgATP驱动的H⁺转运。活跃的H⁺摄取对ATP有绝对需求，呈现简单的米氏动力学（ATP的Km为0.43 mmol/升），希尔系数为0.99。H⁺转运在外部pH为6.7时达到最大值，产生的囊泡内pH为4.8。质子载体如尼日利亚菌素（10⁻⁶ mol/升）或CCCP（10⁻⁵ mol/升）以及非线粒体H⁺ ATP酶抑制剂如NEM或NBD-Cl（浓度为10⁻⁵ mol/升）可消除MgATP依赖性的H⁺积累。线粒体和非线粒体H⁺泵的抑制剂如DCCD（10⁻⁵ mol/升）或Dio 9（0.25 mg/ml）可使微粒体H⁺转运减少约90%。钒酸盐（2×10⁻³ mol/升），一种形成磷酸化中间体的ATP酶的阻滞剂，并不抑制H⁺转运。抑制阴离子转运系统的芪衍生物DIDS（10⁻⁴ mol/升）可完全消除H⁺转运。发现MgATP依赖性的H⁺转运对阴离子有依赖性，顺序为Cl⁻＞Br⁻＞葡萄糖酸盐⁻；在存在SO₄²⁻、CH₃COO⁻或NO₃⁻时，未观察到H⁺转运。MgATP依赖性的H⁺积累对阳离子也有依赖性，顺序为K⁺＞Li⁺＞Na⁺ = 胆碱⁺。如在存在不同离子梯度和离子载体的消散实验所示，在微粒体膜中发现了Cl⁻和K⁺的电导以及少量的H⁺电导。当通过Percoll梯度离心进一步纯化含有H⁺泵的膜时（与匀浆相比富集了9倍），未发现与内质网、线粒体、质膜、酶原颗粒或高尔基体膜的标志物有相关性。目前的数据表明，大鼠外分泌胰腺微粒体中的H⁺泵是一种液泡型或“V”型H⁺ ATP酶，与内质网、高尔基体或内体中描述的H⁺泵最为相似。