RESUMEN
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 â¼60-70% 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 K(M) 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.
Asunto(s)
Glucosa/metabolismo , Hidrógeno/metabolismo , Nitratos/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Transporte Biológico Activo , Activación Enzimática , Espacio Extracelular/metabolismo , HidrólisisRESUMEN
H+ transport driven by V H+-ATPase was found in membrane fractions enriched with ER/PM and Golgi/Golgi-like membranes of Saccharomyces cerevisiae efficiently purified in sucrose density gradient from the vacuolar membranes according to the determination of the respective markers including vacuolar Ca2+-ATPase, Pmc1::HA. Purification of ER from PM by a removal of PM modified with concanavalin A reduced H+ transport activity of P H+-ATPase by more than 75% while that of V H+-ATPase remained unchanged. ER H+ ATPase exhibits higher resistance to bafilomycin (I50=38.4 nM) than Golgi and vacuole pumps (I50=0.18 nM). The ratio between a coupling efficiency of the pumps in ER, membranes heavier than ER, vacuoles and Golgi is 1.0, 2.1, 8.5 and 14 with the highest coupling in the Golgi. The comparative analysis of the initial velocities of H+ transport mediated by V H+-ATPases in the ER, Golgi and vacuole membrane vesicles, and immunoreactivity of the catalytic subunit A and regulatory subunit B further supported the conclusion that V H+-ATPase is the intrinsic enzyme of the yeast ER and Golgi and likely presented by distinct forms and/or selectively regulated.