Functional reconstitution of the Trypanosoma brucei plasma‐membrane D‐glucose transporter

Abstract

The D-glucose transporter of Trypanosoma brucei was solubilized from the plasma membrane and reconstituted into proteoliposomes. Using the reconstitution of D-glucose transport as the assay and non-specific L-glucose uptake as control, we have purified a membrane protein fraction from 7: brucei bloodstream-form ghosts by EDTMalkali treatment and solubilization with the detergents octylglucoside or octylthioglucoside. Upon removal of the detergent by dialysis, the solubilized protein fraction was reconstituted in sonicated liposomes by a freezehaw-sonication step. The reconstituted transporter catalyzed specific D-glucose uptake and was compared in several characteristics with the native facilitated-diffusion transporter as present in live trypanosomes [Seyfang, A. & Duszenko, M. (1991) Eul: J. Biochem. 202, 191-1961. As in vivo, the uptake is time dependent and Na' independent. Transporter substrate affinity and inhibitor specificity are completely retained and it is inhibited by mercuric ions, phloretin and cytochalasin B, but only partially inhibited by phlorizin. The reconstituted transporter also demonstrates trans-stimulation properties indicative of the carrier-mediated transport of D-glucose. In contrast to the human erythrocyte-type glucose transporter, in Z brucei D-fructose uptake was also catalyzed by the same reconstituted protein fraction and specific D-glucose or D-fructose transport were mutually competitive. Both the inhibitor studies and the fructose transport capacity in the reconstituted system are in good agreement with the native transport in live trypanosomes. The specific activity of D-glucose transport was 1.9 5 0.3 nmolmin-'. mg protein-' at 0.2 mM D-glucose and the yield was about 0.8% of total ghost protein after removal of the variant-surface-glycoprotein coat. The successful functional reconstitution of a protozoan glucose transporter represents an important step towards its purification and detailed characterization. This is especially interesting since bloodstream-form trypanosomes depend entirely upon glycolysis for their ATP production.

Murine Models for Trypanosoma brucei gambiense Disease Progression—From Silent to Chronic Infections and Early Brain Tropism

“…T. b. brucei trypanosome fractions were obtained as described previously [35]. Briefly, trypanosomes (about 10 10 cells) were washed in phosphate saline glucose (pH 8.0) at 4°C, centrifuged and hypotonically lysed in 15 ml of 5 mM sodium phosphate, pH 8.0 in the presence of a protease inhibitor cocktail.…”

Phosphatase activity characterization on the surface of intact bloodstream forms ofTrypanosoma brucei

“…Some of the parameters described here were similar to those reported by Seyfang and Duszenko [19], including the acidic nature of the enzyme and the sensitivity to inhibition by tartrate, NaF and vanadate. Other parameters, like the metal interference, could not be discussed because there were no data about metal in that work.…”

“…Phosphatase was purified from cell concentrates as described by Seyfang and Duszenko [19]. Briefly, a ghost fraction was obtained by hypotonic lysis, washed several times and the protein fraction was homogenized in acetate buffer, pH 5.0, and Triton X‐114.…”

A Novel C-terminal Kinesin Is Essential for Maintaining Functional Acidocalcisomes in Trypanosoma brucei

“…Purification Procedure-The fraction corresponding to soluble protein obtained by hypotonic lysis (23) was equilibrated in buffer A (50 mM Tris-HCl, pH 7.0, 20 mM NaCl, 1 mM DTT, 1 mM EDTA, and protease inhibitor mixture) and loaded onto the DEAE fast flow column from Amersham Pharmacia Biotech pre-equilibrated in buffer A. The column was then washed with buffer B, and the flow-through was collected.…”