A spectroscopic assay for the analysis of carbohydrate transport reactions

A carbohydrate-transport assay was developed that does not require isotopic ally labelled substrates, but allows transport reactions to be followed spe ctrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloqu inoline quinone-dependent aldose dehydrogenase [soluble glucose dehydrogena se (sGDH)] and the electron acceptor 2,6-dichloroindophenol (Cl(2)Ind) encl osed in the vesicle lumen. After transport across the vesicular membrane, t he sugar is oxidized by sGDH. The accompanying reduction of Cl(2)Ind result s in a decrease in A(600). The assay was developed and optimized for the la ctose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symp ort and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange tran sport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rate s measured with this assay are comparable with those obtained with the conv entional assay using isotopically labelled substrates. The method is partic ularly suited for determining transport reactions that are not coupled to a ny form of metabolic energy such as uniport reactions, or for characterizin g mutant proteins with a defective energy-coupling mechanism or systems wit h high-affinity constants for sugars.