Solubilization and reconstitution of a chloride transporter from tracheal apical membrane.

Electrogenic Cl- transport in bovine tracheal membrane vesicles was studied by measuring the kinetics of Cl- efflux. Efflux from KCl-loaded vesicles was assayed as the appearance of Cl- in the medium using an anion-specific electrode. Maximal sensitivity was obtained by passage of the vesicles through a Sephadex column immediately before the assay to remove external Cl-. Two components of Cl- transport could be detected. Vesicles exhibited a characteristic spontaneous Cl- efflux that appears to be limited by the cation permeability of the membrane. Addition of the K+-conducting ionophore to increase selectively the permeability to K+ resulted in a stimulation of Cl- efflux. The electrically neutral ionophore nigericin caused only a slight transient increase in Cl- efflux. The valinomycin-dependent flux may be assumed to represent flux through a parallel conductive pathway. Valinomycin-dependent electrogenic Cl- flux was 240 +/- 36 nmol X mg protein-1 X min-1 (n = 4). Soluble extracts obtained with the nonionic detergent octyl glucoside were reconstituted by the freeze-thaw procedure. The reconstituted vesicles exhibited a number of similarities to the native vesicles. Efflux was maximally stimulated with the ionophore valinomycin in the presence of an outwardly directed K+ gradient. Nigericin had no effect on efflux, suggesting the reconstituted transporter is a conductive rather than exchange mechanism. The initial rate of the valinomycin-dependent component was 896 +/- 63 nmol X mg protein-1 X min-1 (n = 5), representing approximately a fourfold increase in specific activity compared with the native membrane vesicle.

Resolution of apical from basolateral membrane of shark rectal gland.

Membrane fractions were isolated from the rectal gland of Squalus acanthias using differential centrifugation and a sucrose gradient run in the presence of 1 M KBr. Using the basolateral membrane marker Na+-K+-ATPase, we obtained a sixfold purification with the most highly purified fraction from the gradient (sp act = 336 +/- 37 mumol X mg protein-1 X h-1). Electrogenic Br- transport was used as a marker activity of the apical membrane, which enabled the identification and purification of a membrane fraction that is highly resolved from the basolateral membrane. The most active fraction was purified approximately 50-fold compared with the crude homogenate. In this fraction, the specific activity of electrogenic anion transport was 296 +/- 87 nmol X mg protein-1 X min-1, whereas the ATPase was only 17.6 +/- 5.7 mumol X mg protein-1 X h-1, representing about a 4-5% contamination of the apical fraction with the basolateral membrane.