Reconstitution of the hexose phosphate translocator from the envelope membranes of wheat endosperm amyloplasts

• Published in: Biochemical journal 1996-11, Vol.319 ( Pt 3) (3), p.717-723
• Main Authors: Tetlow, I J, Bowsher, C G, Emes, M J
• Format: Article
• Language: English
• Subjects: Cell Membrane - metabolism; Dihydroxyacetone Phosphate - metabolism; Glucose-6-Phosphate - metabolism; Glucosephosphates - metabolism; Glyceric Acids - metabolism; Hydrogen-Ion Concentration; Kinetics; Liposomes; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Monosaccharide Transport Proteins - isolation & purification; Monosaccharide Transport Proteins - metabolism; Phosphates - metabolism; Proteolipids - metabolism; Seeds; Triticum - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3190717

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl beta-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (Pi), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/Pi counter-exchange reaction exhibited an apparent K(m) for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (Ki 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/Pi antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.