Separation of tonoplast vesicles enriched in atpase and pyrophosphatase activity from maize roots

Abstract

Continuous sucrose gradient (15–35%) centrifugation of maize (Zea mays L.) root microsomal membranes yielded two well‐separated fractions of tonoplast vesicles located between 19–21% (Peak I) and 25–26% sucrose (Peak II). Marker enzyme analyses indicated that both fractions were essentially free from plasma membrane, mitochondria and Golgi contaminations. The adenosine triphosphate (ATP) supported proton transport activity was found in both Peak I and Peak II with a 70 to 30% distribution. The pyrophosphatase (PP_;) supported proton transport activity was found only in Peak II. Both hydrolytic activities assumed a bell shape pH dependency with pH optimum at 6.5–7.5 and at 6.5–8.5 for ATPase and PP_; ase, respectively. The K_m of the ATPase and PP_iase, at their respective optimal pH, was found to be 1.2 mM and 0.02 mM, respectively. Both ATPase and PPjase activities were strongly inhibited by N.N'‐dicyclohexylcarbodiimide (DCCD) and diethylstilbestrol (DES) but not by molybdate. Peak I contained nitrate‐sensitive and vanadate‐insensitive ATP hydrolysis activity. In addition to catalyzing the nitrate and vanadate‐insensitive hydrolysis of PP; Peak II also contained some minor ATP hydrolysis activity that was sensitive to vanadate and nitrate. The results indicate that H⁺‐ATPases and H⁺‐PP_fase occur different populations of tonoplast vesicles from corn roots.

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