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.

Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes.

“…3 Lower, lanes 3 and 5). Subfractionation of the vesicle preparation by Percoll gradient centrifugation (8) (15,16) or with the electrogenic protonophore CCCP. In the presence of bafilomycin B1 (10 nM) or CCCP (10 ,uM), the MgATP-dependent H+ uptake was abolished (Fig.…”

“…The ADP-ribosylation factor (ARF), a highly conserved low molecular weight GTPbinding protein, has been located in the Golgi complex and implicated to function in intracellular protein transport from the endoplasmic reticulum to and within the Golgi complex (5,6). A monoclonal anti-ARF antibody has been used to detect the ARF protein in a vesicle preparation of rat pancreatic acinar cells containing membranes ofthe endoplasmic reticulum and the Golgi complex (7,8). By immunoelectron microscopy with the same antibody, ARF staining in pancreatic acinar cells was shown to be confined to the cytoplasmic faces of trans-Golgi stack membranes.…”

“…These trans compartments of the Golgi complex have been shown to maintain a low intravesicular pH, which is established by a MgATP-driven vacuolar-type proton pump (9)(10)(11). This vacuolar-type H+-ATPase is present in membrane vesicles from the pancreatic acinar cell preparation (8). Since both ARF (5) and the vacuolar-type H+-ATPase (11) (12) and then electrophoretically transferred to nitrocellulose paper (13,14).…”

“…The pHsensitive dye acridine orange was used to visualize the formation and dissipation of the H+ gradient. H+ uptake was measured as described (8). The difference between the absorbance at 493 and that at 540 nm was recorded in an Aminco DW2 UV/visible spectrophotometer.…”

Structural and functional aspects of calcium homeostasis in eukaryotic cells

“…Schultz and coworkers [287,2881 have recently suggested that Ca2+ accumulation by the Ins(l,4,5)P3-sensitive store does not depend on the activity of a Caz+-ATPase, but rather on a more complicated mechanism involving first the formation of a pH gradient (acidic interior), due to a H+-ATPase and subsequently an electro-neutral exchange of 2 H ' ,'Ca2+.…”