Pulse-labeling experiments in transmembrane transport studies

Many sugars, when added to the medium of bacteria or yeast cells, are recovered inside the cell partly as the sugar-6-phosphate and partly as the free sugar. Phosphorylation may have occurred intracellularly subsequent to transmembrane transport of the free sugar, or during transport, intimately coupled to the translocation step itself. When using nonmetabolizable sugars, isotope pulse-labeling experiments can be used to discriminate between these two possibilities. In previous papers these pulse-labeling procedures have been discussed and interpreted only on a qualitative basis. Due to experimental or systematic errors—such as adsorption of labeled substrate on the filters used to separate cells and medium—the interpretation is not always unambiguous. Under these circumstances a more detailed quantitative analysis of the kinetics of pulse-labeling could provide a warrant for the reliability of the interpretation.With non-metabolizable sugars a stationary state will usually develop, characterized by a dynamic equilibrium between the free sugar and the sugar-phosphate. In the present paper the kinetics of pulse-labeling during this stationary state are derived.