Solubization of Na/K/Cl Cotransporter in Rabbit Parotid Acini and Its Purification-Separation of Bumetanide Binding Protein by ^(14)C-NEM Labeling

Abstract

In rat parotid basolateral membranes the presence of a 160 kD protein can be labeled with the irreversible sulfhydryl reagent [^(14)C]-N-ethyl maleimide in a bumetainide-proectable fasion. The previous results suggest that the existence of an esential sulfhydryl group is closly associated with the bumetanied-binding site on the parotid Na/K/Cl cotransporter, provide strong evidence that this protein is a part or all of the parotid bumetanide-binding site. When this protein is treated with endoglycosidase F/N-glycosidase F to remove N linked oligosaccharides, its apparent molecular weight decreases to 135 kD. The bumetanide-binding protein was purified using two preparative electrophoresis steps. First, a Triton X-100 extraet enriched in this protein was run on preparative electrophoresis to obtain fractions containing proteins in the 160 kD range. These were then deglycosylated with endoglycosidase F/N-glycosidase F and selected fractions were pooled and rerun on preparative electrophoresis to obtain a final 135 kD fraction. The enrichment of the bumetanide-binding protein in this final 135 kD fraction estimated from [^(14)C]-N-ethylmaleimide labeling was approximately 48 times relative to the starting membrane extract. Since the bumetanide-binding site represents approximately 2% of the total protein this starting extract, this enrichment indicates a high degree of purity of this protein in the 135 kD fradion.