Filter assay technique and quench-flow experiments: examples of receptor-mediated transmembrane ion-exchange measured with membrane vesicles.
Modifications to a quench-flow apparatus are described which allow a rapid, in-line filter assay with immediate washing, in conditions to give minimum background. A design for an effluent spout is presented, which decelerates the liquid by a large factor, prevents splashes, limits the area of the filter exposed to the sample and allows an immediate wash over a larger area. A design for a filter assay funnel for general use is also presented. These devices feature minimal contact of the funnels with the filter disc. Examples are given in which in-line filtration was used to follow transmembrane ion flux in membrane vesicle preparations. In measurements of transmembrane flux with membrane vesicles and radioisotope the filter assay background can be resolved into three components. These are, (1) the uptake of radioactivity by the filter, (2) the radioactivity inside the vesicles not taking part in the specific measurement and (3) the occlusion of radioactivity in aggregated membrane particles on the filter. These different components depend on the conditions in different ways. Techniques for minimizing the background in filter assays are discussed. The importance of rapid filtration and immediate washing is demonstrated. The examples given illustrate that the function of the acetylcholine receptor from E. electricus is not affected by diisopropylfluorophosphate in the conditions used, and that added GABA is not removed from solution in a brain membrane preparation by the GABA uptake mechanisms in the short times of the experiments.
Cash
D J
DJ
Department of Biochemsitry, University of Missouri-Columbia 65211.
Subbarao
K
K
Bradbury
J R
JR
Mayes
G M
GM
D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D004593:Electrophorus / Q000378:metabolism; D005374:Filtration / Q000295:instrumentation / Q000379:methods*; D007474:Ion Exchange; D008566:Membranes / Q000378:metabolism; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism; D011963:Receptors, GABA-A / Q000378:metabolism; D017981:Receptors, Neurotransmitter / Q000378:metabolism*
D011950:Receptors, Cholinergic; D011963:Receptors, GABA-A; D017981:Receptors, Neurotransmitter