IDENTIFICATION, CHARACTERIZATION AND PURIFICATION OF A 160 KD BUMETANIDE-BINDING GLYCOPROTEIN FROM THE RABBIT PAROTID

We demonstrate the presence of a 160 kD protein in rabbit parotid baso lateral membranes that can be labeled with the irreversible sulfhydryl reagent [C-14]-N-ethylmaleimide in a bumetanide-protectable fashion. The specificity of this labeling, and our previous evidence for the ex istence of an essential sulfhydryl group closely associated with the ( bumetanide-binding site on the parotid Na+-K+-Cl- cotransporter J. Mem brane Biol. 112:51-58, 1989), provide strong evidence that this protei n is a part or all of the parotid bumetanide-binding site. When this p rotein is treated with endoglycosidase F/N-glycosidase F to remove N-l inked oligosaccharides, its apparent molecular weight decreases to 135 kD. The pl of this deglycosylated protein is almost-equal-to 6.4. The bumetanide-binding protein was purified using two preparative electro phoresis steps. First, a Triton X-100 extract enriched in this protein was run on preparative electrophoresis to obtain fractions containing proteins in the 160 kD range. These were then deglycosylated with end oglycosidase 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 k D fraction estimated from [C-14]-N-ethylmaleimide labeling was approxi mately 48 times relative to the starting membrane extract. Since the b umetanide-binding site represents approximately 2% of the total protei n in this starting extract, this enrichment indicates a high degree of purity of this protein in the 135 kD fraction.