Preparation of sealed Torpedo marmorata membrane fragments suitable for quantitative tracer flux studies

Sealed vesicular membrane fragments (microsacs) prepared from Torpedo electric organs have been used extensively in tracer flux experiments. Microsac suspensions with high AcChR and low AcChE content are obtained by density gradient separation of crude electric organ homogenates [I-S]. Special methods selectively yielding gradient fractions containing microsacs with functionally intact AcChR have been developed [6,7]. However, recent advances in the techniques [8-lo] and in the mathematical analysis [ 11,121 of flux measurements, raise the need for more suitable methods of preparation. selective neuroactivator-induced filling (AcCh pulses) of functional microsacs with Li’; (iii) Separation of Li’-filled and Cs’-filled microsacs using a continuous self-generating Percoll gradient; (iv) Extensive characterization of the resulting fractions on the basis of stringent criteria.