Rapid fluorescence assay of glucose and neutral solute transport using an entrapped volume indicator.
A rapid fluorescence method is described for quantitative measurement of glucose and neutral solute transport in cells and sealed membrane vesicles based on volume changes accompanying transport. Membrane vesicles are loaded with the volume-sensitive indicator fluorescein sulfonate (FS) which undergoes concentration-dependent fluorescence self-quenching. In response to an inward solute gradient, there is a rapid decrease in vesicle volume due to osmotic water efflux, followed by solute entry and volume increase. Rates of solute influx are calculated from the time course of vesicle volume reported by FS fluorescence. To validate the method, D-glucose transport was measured in sealed red cell ghost membranes (RBC) and microvillus vesicles (MVV) isolated from human placenta. RBC D-glucose transport was stereospecific and inhibited by cytochalasin B (KI = 0.2 microM) and phloretin (KI = 4 microM). D-Glucose fluxes determined by FS fluorescence were identical to those determined by D-[3H]glucose uptake; in D-[3H]glucose uptake studies, FS did not itself alter D-glucose transport. In MVV, D-glucose transport was saturable (Km = 25 +/- 1 mM, Vmax = 8 +/- 1 nmol/s.mg protein) and inhibited by cytochalasin B and phloretin. This fluorescence transport assay provides an accurate method to measure neutral solute uptake in sealed membranes (cells, vesicles liposomes), of particular use for rapid screening of large numbers of samples, when labeled solute is unavailable, or when the quantity of membrane or isolated transport protein is limited.
Kim
Y K
YK
Department of Medicine, University of California, San Francisco 94143.
Illsley
N P
NP
Verkman
A S
AS
D001692:Biological Transport; D001786:Blood Glucose / Q000378:metabolism; D002462:Cell Membrane; D002463:Cell Membrane Permeability; D004910:Erythrocyte Membrane; D004912:Erythrocytes / Q000378:metabolism; D005452:Fluoresceins; D005470:Fluorometry; D006801:Humans; D010920:Placenta
D001786:Blood Glucose; D005452:Fluoresceins