Abstract
V H+-ATPase has an important role in a variety of key physiological processes. This enzyme is reversibly activated/partly inactivated by the addition/exhaustion of extracellular glucose. The current model of its regulation assumes the reversible disassembly/reassembly of ∼60–70% of the V1 and V0 membrane complexes, which are responsible for ATP hydrolysis and H+ conductance, respectively. The number of assembled complexes determines the pump activity because disassembled complexes are inactive. The model predicts the identical catalytic properties for the activated and semi-active enzymes molecules. To verify the model predictions we have isolated total membranes from yeast spheroplasts that were pre-incubated either with or without glucose. Nitrate treatment of membranes revealed the similar ATPase inhibition for two enzyme states, suggesting that they have identical structures that are essential for ATP hydrolysis. However, H+ transport was inhibited more than the ATPase activities, indicating a nitrate uncoupling action, which was significantly higher for the nonactivated enzyme. This finding suggests that the structure of the non-activated enzyme, which is essential for H+ transport, is less stable than that of the activated enzyme. Moreover, the glucose activation of the pump increases i) its coupling capacity; ii) its KM for ATP hydrolysis and ATP affinity for H+ transport; iii) the Vmax for H+ transport in comparison with the Vmax for ATP hydrolysis and iv) the immune reactivity of catalytic subunit A and regulatory subunit B by 9.3 and 2.4 times, respectively. The protein content of subunits A and B was not changed by extracellular glucose. We propose that instead of the dissociation/reassociation of complexes V1 and V0, changes in the extracellular glucose concentration cause reversible and asymmetrical modulations in the immune reactivity of subunits A and B by their putative biochemical modifications. This response asymmetrically modulates H+-transport and ATP hydrolysis, exhibiting distinct properties for the activated versus non-activated enzymes.
Highlights
The V H+-ATPases pump H+ from the cytosol across membranes for a variety of intracellular organelles, acidifying their lumen, and across the plasma membranes of specialised cells [1,2,3,4,5,6,7,8,9,10]
The modulation of its activity by extracellular glucose is similar to that found for the P H+-ATPase of the yeast Saccharomyces cerevisiae [2,7,9,10,17,18,19,20,21,22]
Because the enzyme shows ATPase activity only when the V1 complex is bound with the V0 complex and the membrane, our data suggest that both enzyme states display similar binding and/or interactions between the subunits of the V1 complex and its binding with the membrane
Summary
The V H+-ATPases pump H+ from the cytosol across membranes for a variety of intracellular organelles, acidifying their lumen, and across the plasma membranes of specialised cells [1,2,3,4,5,6,7,8,9,10]. This difference between the two enzyme states is greater when comparing the ratios of the initial velocities of H+ transport by activated and non-activated enzymes with increasing nitrate concentrations (Fig. 2, insert).
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