Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site

Abstract

We demonstrate that the high affinity bumetanide binding site of the rabbit parotid acinar cell can be extracted from a basolateral membrane fraction using relatively low concentrations (0.07%, wt/vol; 1 mg membrane protein/ml) of the nonionic detergent Triton X-100. This extracted site cannot be sedimented by ultracentrifugation at 100,000 x g x 1 hr. Bumetanide binding to this site retains the ionic characteristics of bumetanide binding to native membranes but shows a fivefold increase in binding affinity (Kd = 0.57 +/- 0.15 microM vs. Kd = 3.3 +/- 0.7 microM for native membranes). Inactivation of the extracted bumetanide binding site observed at detergent/protein ratios greater than 1 can be prevented or (partially) reversed by the addition of exogenous lipid (0.2% soybean phosphatidylcholine). When the 0.07% Triton extract is fractionated by sucrose density gradient centrifugation in 0.24% Triton X-100, 0.2% exogenous lipid and 200 mM salt, the high affinity bumetanide binding site sediments as a single band with S20,w = 8.8 +/- 0.8 S. This corresponds to a molecular weight approximately 200 kDa for the bumetanide binding protein-detergent-lipid complex and represents a sevenfold purification of this site relative to the starting membrane fraction. In contrast to previous attempts to purify Na/K/Cl cotransport proteins and their associated bumetanide binding sites, the present method avoids harsh detergent treatment as well as direct covalent modification (inactivation) of the transporter itself. As a consequence, one can follow the still active protein through a series of extraction and purification steps by directly monitoring its bumetanide binding properties.

Ca(2+)‐dependent heat production by rat skeletal muscle in hypertonic media depends on Na(+)‐Cl‐ co‐transport stimulation.

“…However, as already pointed out, this co-transport can only continue, during the hyperosmotic steady state, thanks to the sustained stimulation of active Na+-K+ transport due to the increase of intracellular Na+ ion concentration. Now it is evident that none of the usual criteria by which Na+-K+-Cl-co-transport is defined -86Rb+ uptake in presence and absence of bumetanide (Burnham, Kidd & Palfrey, 1990); dependency on extracellular K+ (Aickin, Betz & Harris, 1989); bumetanide binding site solubilization and purification studies (Turner & George, 1990) -allows one to unambiguously exclude that this three-ion system simply results from a combination of Na-Cl-co-transport and active Na+-K+ transport. In fact, some close functional association of Na+-Cl-co-transport, which is a secondary-active transport process, with the primary-active Na+-K+ transport system is even suggested by the finding, in two non-muscle cell types, that the Na+-K+-Cl-co-transport system is dependent upon intracellular ATP concentration (Homma, Burns & Harris, 1990;Flatman, 1991).…”

Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters

“…After the discovery of electrically silent cotransport mechanisms in mammalian cells and tissues 25 years ago (138,340), several laboratories undertook unrewarded attempts to identify the proteins responsible for such a transport system (94,107,121,197,251,406). However, the major breakthrough in this field came in the early 1990s from two different laboratories that were able to identify the genes responsible for Na ϩ -Cl Ϫ (136, 137) and Na ϩ -K ϩ -2Cl Ϫ cotransporters (136,311,440).…”

Involvement of Direct Phosphorylation in the Regulation of the Rat Parotid Na+-K+-2Cl− Cotransporter

“…Ϫ cotransporter could be preserved in detergent solutions by the addition of suitable exogenous lipids (16,17). The procedure was as follows.…”

“…Binding Assay-Equilibrium bumetanide binding was measured using a nitrocellulose filtration assay as described previously (16,18). Briefly, a 20-l aliquot of sample was combined with 20 l of incubation medium consisting of either Buffer K containing 10 Ci/ml [ 3 H]bumetanide or the same medium with all potassium replaced by Na ϩ .…”

“…After a 15-min incubation the reaction was terminated by the addition of ice-cold stop solution followed by Millipore filtration (HAWP 0.45 m). Other procedures were as described previously (16,18). [ 3 H]bumetanide binding observed in the absence of Na ϩ was subtracted from that observed in its presence to yield the Na ϩ -dependent component of binding.…”