Characterization in tobacco leaves of structurally and functionally different membrane fractions enriched in vanadate sensitive H +-ATPase

Abstract

A membrane fraction enriched in vanadate-sensitive H +-ATPase was obtained from tobacco leaves ( Nicotiana tabacum L. cv. Xanthi) by phase partitioning. Subfractions were then prepared by counter-current distribution. All fractions contained an H +-ATPase activity with similar characteristics. Fractions differed however by their sidedness. In addition, in the presence of lysophosphatidylcholine, the right-side-out enriched fraction exhibited an ATPase activity three-times higher than the inside-out enriched fraction. This difference was shown not to be due to selective solubilization of various subfractions by the detergent nor to specific effect of detergent on kinetic parameters of the H +-ATPase when vesicles were solubilized. Immunological determination of the H +-ATPase content, by ELISA, showed that subfractions differed by the amount of the enzyme present in the vesicles. Accordingly, differences in ATP hydrolysis activity were shown to be related to differences in ATPase content. In addition, the proton translocation activity of one of the subfractions exhibited a sensitivity to auxin 100-times higher than that of other fractions. Finally, an analysis by 2-D electrophoresis and quantitative gel imaging was performed. Polypeptide patterns of various subfractions appeared to differ for a lot of polypeptides by factors ranging from +200% to +500%. Taken together these results show that the counter-current distribution method allows one to separate tobacco leaf membrane subfractions which all possess a vanadate-sensitive H +-ATPase as the nearly only ATPase activity, but are structurally and functionally different.