An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins.
A new method for the measurement of rapid isotopic release from a membrane compartment is described. Membrane vesicles loaded with isotope, or broken membranes with bound radioactive ligand, are filtered onto the surface of a cellulose ester filter; the rate of the loss of isotope from the membrane compartment is followed continuously by collecting fluid which is passed through the filters under high pressure. A change in release rate is initiated by changing the solution or by delivering a flash of light to a photosensitive sample. The approach has been used to study rapid 22Na efflux from membrane vesicles rich in the ouabain-sensitive Na pump, and to examine dissociation of 32P and 86Rb from membrane-bound Na,K-ATPase. Since the rate of efflux is measured, and not the total counts remaining on the filter, the technique has high sensitivity. A complete time course is obtained using only a few micrograms of membrane protein. The apparatus described is simple, inexpensive, and easily constructed; with the present device, time resolution is approximately 10 ms.
D000818:Animals; D004285:Dogs; D005374:Filtration / Q000379:methods; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008024:Ligands / Q000032:analysis*; D008565:Membrane Proteins / Q000032:analysis*; D010777:Photochemistry; D011312:Pressure; D011868:Radioisotopes / Q000032:analysis*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis
D008024:Ligands; D008565:Membrane Proteins; D011868:Radioisotopes; D000254:Sodium-Potassium-Exchanging ATPase