A spectroscopic assay for the analysis of carbohydrate transport reactions.
A carbohydrate-transport assay was developed that does not require isotopically labelled substrates, but allows transport reactions to be followed spectrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloquinoline quinone-dependent aldose dehydrogenase [soluble glucose dehydrogenase (sGDH)] and the electron acceptor 2,6-dichloroindophenol (Cl2Ind) enclosed in the vesicle lumen. After transport across the vesicular membrane, the sugar is oxidized by sGDH. The accompanying reduction of Cl2Ind results in a decrease in A600. The assay was developed and optimized for the lactose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symport and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange transport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rates measured with this assay are comparable with those obtained with the conventional assay using isotopically labelled substrates. The method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy-coupling mechanism or systems with high-affinity constants for sugars.
Heuberger
E H
EH
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology, University of Groningen, The Netherlands.
Poolman
B
B
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D001692:Biological Transport; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins; D042843:Glucose 1-Dehydrogenase; D005948:Glucose Dehydrogenases / Q000378:metabolism; D007553:Isotope Labeling; D007700:Kinetics; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008756:Methylgalactosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010084:Oxidation-Reduction; D011510:Proteolipids / Q000378:metabolism; D018892:Proton-Motive Force; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D013053:Spectrophotometry / Q000379:methods; D013291:Streptococcus; D013379:Substrate Specificity; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D011510:Proteolipids; D027981:Symporters; D013862:Thiogalactosides; C020485:proteoliposomes; C009468:thiomethylgalactoside; C021499:lactose permease; D015086:2,6-Dichloroindophenol; D002237:Carbohydrate Dehydrogenases; D005948:Glucose Dehydrogenases; D042843:Glucose 1-Dehydrogenase; C082601:aldose dehydrogenase (pyrroloquinoline-quinone)