6533b81ffe1ef96bd1278855

RESEARCH PRODUCT

Active vanadate-sensitive H+ translocation in corn roots membrane vesicles and proteoliposomes

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description

Abstract A member fraction from corn roots which contains a vanadate-sensitive ATPase activity has been prepared. The specific activity at 38°C is between 3 and mol 12 μmol · min −1 · mg −1 , depending on the age of roots. Addition of ATP promotes a very rapid quenching of the fluorescence of 9-amino-6-chloro-3-methoxy-acridin (ACMA). Proton pumping exhibits a delayed sensitivity to vanadate but is strongly and instantaneously inhibited by the new inhibitor SW 26. Both proton pumping, measured by the initial quenching rate, and ATP hydrolysis show maximum activities at ATP concentrations in the millimolar range, but the apparent K m -value for hydrolysis is higher than that observed for proton pumping. This is interpreted as being due to the presence of two populations of ATPases, one of them hydrolyzing ATP without creating a pH-gradient. The vanadate-sensitive ATP hydrolysis and H + -pumping activity may be solubilized with lysolecithin and reconstituted into liposomes either by a freeze-thawing-sonication or an octylglucoside dilution procedure. Both methods yield proteoliposomes exhibiting very effecient proton pumping, which is more sensitive to vanadate (I 50 = 2 μ M) or to SW 26 (I 50 = 0.5 μ M) than that of the original membrane fractions.