Open AccessPurification of the lactose:H + carrier of Escherichia coli and characterization of galactoside binding and transport
Author(s) -
WRIGHT J. Keith,
OVERATH Peter
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb07944.x
Subject(s) - galactoside , chemistry , vesicle , substrate (aquarium) , membrane , symporter , chromatography , electrochemical gradient , lactose permease , biochemistry , membrane transport protein , membrane protein , enzyme , biology , transporter , gene , ecology
The lactose carrier, a galactoside:H + symporter in Escherichia coli , has been purified from cytoplasmic membranes by pre‐extraction of the membranes with 5‐sulfosalicylate, solubilization in dodecyl‐ O ‐β‐ d ‐maltoside, Ecteola‐column chromatography, and removal of residual impurities by anti‐impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles. Purification was monitored by tracer N ‐[ 3 H]ethylmaleimide‐labeled carrier and by binding of the substrate p ‐nitrophenyl‐α‐ d ‐galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria. Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain. Large unilamellar proteoliposomes (1–5‐μm diameter) were prepared from initially small reconstituted vesicles by freeze‐thaw cycles and low‐speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (Δp H+ ) or one of its components (ΔΨ or ΔpH). Comparison of the steady‐state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport. The half‐saturation constants for active uptake of lactose ( K T = 200μM) or β‐ d ‐galactosyl‐1‐thio‐β‐ d ‐galactoside ( K T = 50–80 μM) by the purified carrier were found to be similar to those measured in cells or cytoplasmic membrane vesicles. The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles ( k cat = 3–4s −1 for lactose) but considerably smaller than in cells ( k cat = 40–60s −1 ). Possible reasons for this discrepancy are discussed.