Surcose transport in isolated plasma-membrane vesicles from sugar beet (Beta vulgarisL.) Evidence for an electrogenic sucrose-proton symport
✍ Scribed by Thomas J. Buckhout
- Book ID
- 104752727
- Publisher
- Springer-Verlag
- Year
- 1989
- Tongue
- English
- Weight
- 776 KB
- Volume
- 178
- Category
- Article
- ISSN
- 0032-0935
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✦ Synopsis
An analysis of the molecular mechanism of sucrose transport across the plasmalemma was conducted with isolated plasma-membrane (PM) vesicles. Plasma membrane was isolated by aqueous two-phase partitioning from fully expanded sugar beet (Beta vulgaris L.) leaves. The isolated fraction was predominantly PM vesicles as determined by marker-enzyme analysis, and the vesicles were oriented right-side-out as determined by structurally linked latency of the PM enzyme, vanadate-sensitive Mg 2 +-ATPase. Sucrose uptake was investigated by equilibrating PM vesicles in pH 7.6 buffer and diluting them 20-fold into pH 6.0 buffer. Using this pH-jump technique, vesicles accumulated acetate in a pH-dependent, protonophore-sensitive manner, which demonstrated the presence of a pH gradient (ApH) across the vesicle membrane. Addition of sucrose to pH-jumped PM vesicles resulted in a pH-dependent, protonophoresensitive uptake of sucrose into the vesicles. Uptake was sucrose-specific in that a 10-fold excess ofmannose, glucose, fructose, mannitol, melibiose, lactose or maltose did not inhibit sucrose accumulation. The rate of pH-dependent uptake was saturable with respect of sucrose concentration and had an apparent Km of 0.45 mM. Sucrose uptake was stimulated approximately twofold by the addition of valinomycin and K +, which indicated an electrogenic sucrose-H + symport. Membrane potentials (A 0) were imposed across the vesicle membrane using valinomycin and K +. A membrane potential, negative inside, stimulated pH-dependent sucrose uptake while a AO, positive inside, Abbreviations and symbols: CCCP = carbonyl cyanide m-chlorophenylhydrazone; cyt = cytochrome; PM = plasma-membrane(s); AO = electrical potential difference The mention of vendor or product does not imply that they are endorsed or recommended by U.S. Department of Agriculture over vendors of similar products not mentioned inhibited uptake. Conditions that produce a negative A 0 in the absence of a pH gradient supported, although weakly, sucrose uptake. These data support an electrogenic sucrose-H + symport as the mechanism of sucrose transport across the PM in Beta leaves.