Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site.
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.
Turner
R J
RJ
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
George
J N
JN
D000818:Animals; D001665:Binding Sites; D002034:Bumetanide / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification; D002499:Centrifugation, Density Gradient; D002712:Chlorides / Q000378:metabolism; D004232:Diuretics / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification; D017830:Octoxynol; D010306:Parotid Gland / Q000378:metabolism*; D011092:Polyethylene Glycols; D011188:Potassium / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D012995:Solubility
D002352:Carrier Proteins; D002712:Chlorides; D004232:Diuretics; D008565:Membrane Proteins; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D011092:Polyethylene Glycols; D017830:Octoxynol; D012964:Sodium; D011188:Potassium