A rapid-filtration technique for membrane fragments or immobilized enzymes: measurements of substrate binding or ion fluxes with a few-millisecond time resolution

Abstract

The construction and use of a filtration system with milliseconds time resolution is described here. This apparatus allows measurements of substrate binding to immobilized enzyme or ion fluxes through membrane vesicles to be performed over a very large time scale, from 10 ms to seconds. The main advantage of this system compared to the widely used quench-flow technique is that it does not require the use of an inhibitor. Following adsorption of the enzyme in an adequately chosen filter, the reaction is allowed to proceed within the filter during a forced filtration of a buffer containing the reactive substrate (or of a washing solution in the case of efflux measurements). The design allows the duration of filtration and buffer flux to be finely and reproducibly controlled. This paper illustrates the use of this rapid-filtration system for time-resolved measurements of calcium binding and transport by sarcoplasmic reticulum Ca-ATPase and of the initial phase of ADP transport by the ADP/ATP carrier of intact mitochondria.