Reconstitution and Characterization of H+-Translocating ATPase from the Plasma Membrane of Phaseolus mungo L. Roots |
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Authors: | Kasamo Kunihiro |
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Affiliation: | Department of Cell Biology, National Institute of Agrobiological Resources Tsukuba Science City, Yatabe, Ibaraki 305, Japan |
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Abstract: | Plasma membrane H+-translocating ATPase was partially purifiedfrom mung bean (Phaseolus mungo L.) roots and reconstitutedinto soybean phospholipid (asolectin) liposomes by the n-octylglucosidedilution method. The resulting proteoliposomes were mainly unilamellarvesicles ranging in size from 0.05 to 0.2 µm. The existenceof ATP-drived H+-pumping across the proteoliposomes was demonstratedby the quenching of quinacrine fluorescence in the presenceof Mg2+. The quenching could be abolished by an uncoupler, FCCP,and an inhibitor of H+-translocating ATPase, vanadate. The reconstitutedATPase consisted of three major polypeptides of 105 KDa, 67KDa and 57 KDa. Its pH optimum, divalent cation stimulationand vanadate sensitivity were similar to those of partiallypurified ATPase. However, the specificity toward ATP was muchgreater following reconstitution. Also reconstitution reducedthe degree of inhibition by DCCD. Local anesthetics (e.g. dibucaine)had no effect on H+-pumping activity but increased the ATPaseactivity when proteoliposomes were reconstituted in their presence. (Received May 2, 1986; Accepted October 17, 1986) |
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