A Computer Controlled Device to Facilitate Studies of the Kinetics of Ligand-Binding: Binding of Diazepam to Bovine Brain Membranes

Abstract

A device to facilitate kinetic receptor filtration assays is described. The receptor containing membranes and the labeled ligand are placed in two separate syringes and are rapidly mixed into a collecting syringe using a pneumatic ram. Shortly after the start of mixing, a pneumatically controlled valve switches the collecting syringe containing the receptor-ligand mixture to the filtration unit. Filtration is performed on glass/microfiber filters or equivalent by pushing the plunger of the collecting syringe by a stepper motor. A valve positioner controlling several valves allows the filtered membranes to be washed and dried by pressure in any user programmable sequence. Further filtration of the receptor-ligand mixture can be programmed at selected time points. The entire system is controlled by an IBM-PC. With this system, the association and dissociation of diazepam from crude bovine-brain membranes has been studied at 4 °C. The dissociation shows a biphasic pattern with half lifes of 1.3 and more than 23 minutes respectively. Association appears to be into a single compartment.