title: Affinity purification of sucrose binding proteins from the plant plasma membrane.
abstract: Purified plasma membranes from sugar beet leaves were solubilized by 1% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate and loaded on a sepharose 6 B column substituted with sucrose. Elution with sucrose at pH 5.2 yielded a peak that represented 0.2% of the loaded protein. This peak did not appear when the samples were pretreated with either 0.5 mM N-ethylmaleimide (NEM) or 0.5 mM para-chloromercuribenzenesulfonic acid. It was also absent when palatinose, a sucrose analogue not recognized by the sucrose transporter, was used as the affinity ligand. The peak specifically eluted by sucrose from the sucrose-Sepharose column exhibited sucrose transport activity after reconstitution into proteoliposomes. This peak was further fractionated by ion-exchange chromatography on a Mono-Q column, and the different fractions obtained were differentially labeled by [3H]NEM in the presence of sugars recognized (sucrose, maltose) or not recognized (palatinose) by the sucrose transporter. The data allowed to identify two fractions that were enriched with two polypeptides (56 and 41 kDa) differentially labeled by NEM in the presence of sucrose.
authors.affiliation: UA CNRS 574, Laboratoire de Biologie et Physiologie Végétales, Université de Poitiers, France.
authors.forename: Z S
authors.initials: ZS
authors.lastname: Li
authors.forename: A M
authors.initials: AM
authors.lastname: Noubhani
authors.forename: A
authors.initials: A
authors.lastname: Bourbouloux
authors.forename: S
authors.initials: S
authors.lastname: Delrot
mesh_terms: D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry*; D002846:Chromatography, Affinity; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D010940:Plant Proteins; D013395:Sucrose