Kinetics analysis of the plasma membrane sucrose-H+ symporter from sugar beet (Beta vulgaris L.) leaves

Abstract

The kinetics behavior of the H+-sucrose (Suc) symporter was investigated in plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves by analyzing the effect of external and internal pH (pHo and pHi, respectively) on Suc uptake. The apparent Km for Sue uptake increased 18-fold as the pH0 increased from 5.5 to 7.5. Over this same pHo range, the apparent Vmax for Sue uptake remained constant. The effects of pHi in the presence or absence of internal Sue were exclusively restricted to changes in Vmax. Thus, proton concentration on the inside of the membrane vesicles ([H+]1) behaved as a noncompetitive inhibitor of Suc uptake. The Km for the proton concentration on the outside of the membrane vesicles was estimated to be pH 6.3, which would indicate that at physiological apoplastic pH Sue transport might be sensitive to changes in pH0. On the other hand, the [H+]; for half-maximal inhibition of Suc uptake was approximately pH 5.4, making regulation of Sue transport through changes in [H+]i unlikely. These results were interpreted in the framework of the kinetics models for co-transport systems developed by D. Sanders, U.-P. Hansen, D. Gradmann, and C. L. Slayman (J Membr Biol [1984] 77: 123-152). Based on their analysis, the behavior of the Suc symporter with respect to the [H+]i is interpreted as an ordered binding mechanism by which the binding of Sue on the apoplastic side of the membrane and its release on the symplastic side precedes that of H+ (i.e. a first-on, first-off model).