Solubilization of Na/K/Cl Cotransporter in Rabbit Parotid Acini and Its Purification-Purification by Sucrose Density Gradient

Abstract

The high affinity bumetanide binding site of the rabbit parotid acinar cell can be extracted from a basolateral membrane fraction using rotative low concentration (0.07%, 1 ㎎ membrane protein/㎖) of the nonionic detergent Triton x-100. This extracted site can not be sedimented by uleracentrifugation at 100,000×g×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. Inactivation of the extracted bumetanide binding site observed at detergent/protein ratios>1 can be partially prevented by the addition of 0.2% soubeen 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 bumetanide binding site sedtments as a singlle band with S_(20,w)=8.8±0.8 S. This corresponds to a molecuaar weight -200 kD for the bumetanide binding protein-detergent-lipid complex and represents a sevenfold purifiratioa of this site relative to the starting membrane fraction. Consequrntly, it may be suggested that partial purification of bumetanide binding site ran be able to obtained using the 0.07% Triton X-100 and sucrose density gradient centrifugation.