Characterization of MgATP-Driven H+ uptake in to a microsomal vesicle franction from rat pancreatic acinar cells

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.