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Characterization of MgATP-driven H+ uptake into a microsomal vesicle fraction from rat pancreatic acinar cells
F Thévenod1, T P Kemmer, A L Christian
1Max-Planck-Institut für Biophysik, Frankfurt/Main, Federal Republic of Germany.
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.
Insights
This study identifies a magnesium-ATP-dependent proton (H+) pump in rat exocrine pancreas microsomes. This pump functions as a vacuolar-type H+ ATPase, crucial for cellular transport processes.
Area of Science:
- Cell Biology
- Biochemistry
- Physiology
Background:
- Microsomal vesicles from exocrine pancreas cells are involved in various cellular transport functions.
- Understanding the mechanisms of proton (H+) transport is essential for cellular homeostasis and function.
Purpose of the Study:
- To characterize the MgATP-driven H+ transport system in rat exocrine pancreas microsomes.
- To identify the type and properties of the H+ pump involved.
Main Methods:
- Measurement of H+ transport via acridine orange accumulation in isolated microsomal vesicles.
- Kinetic analysis of ATP dependence and substrate specificity.
- Inhibition studies using protonophores, ATPase inhibitors, and anion transport blockers.
- Ion dependency studies (cations and anions).
- Membrane purification using Percoll gradient centrifugation.
Main Results:
- MgATP-driven H+ transport exhibited Michaelis-Menten kinetics with a Km for ATP of 0.43 mmol/liter.
- H+ transport was optimal at pH 6.7, generating an intravesicular pH of 4.8.
- The pump was sensitive to inhibitors of non-mitochondrial H+ ATPases and anion transport, but not vanadate.
- Transport was dependent on specific anions (Cl- > Br-) and cations (K+ > Li+).
- Purified membranes showed no correlation with other organelle markers, suggesting a unique microsomal localization.
Conclusions:
- The identified H+ pump is a vacuolar- or "V"-type H+ ATPase.
- This V-type H+ ATPase shares similarities with those found in endoplasmic reticulum, Golgi apparatus, and endosomes.
- The findings provide insights into the proton transport mechanisms within pancreatic exocrine cells.
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