Characterization of the pyrophosphate‐dependent proton transport in microsomal membranes from maize roots

Cleared maize ( Zea mays L. cv. LG 11) root homogenates were prepared and layered on the top of sucrose step gradients (10, 35 and 45%). The ATP‐ and pyrophosphate (PP i )‐dependent proton‐pumping activities were recovered almost completely at the 10%/35% interface, corresponding to the microsomal fraction (Golgi, tonoplast and endoplasmic reticulum). The PP i ‐dependent proton pump was characterized by the fluorescence quenching of quenching of quinacrine. The pH optimum was 7 to 8. The H + ‐PPase was Mg 2+ ‐dependent and the K m for PP i (in the presence of 3 m M MgSO 4 ) was 28 μ M . The pump was electrogenic, K + ‐dependent and a permeant anion was necessary to dissipate the membrane potential (NO 3 − = I − >Br − > Cl − ). No activity was detected in the presence of electroneutral proton inonophores or, when valinomycin was added, with electrogenic ionophores. The H + ‐PPase was insensitive to vanadate, oligomycin and molybdate. ‐Diethylstilbestrol (DES) and N,N′‐dicyclohexylcarbodiimide (DCCD) were strongly inhibitory at 100 μ M .