An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins
✍ Scribed by Bliss Forbush III
- Publisher
- Elsevier Science
- Year
- 1984
- Tongue
- English
- Weight
- 970 KB
- Volume
- 140
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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.