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 basolateral membranes that can be labeled with the irreversible sulfhydryl reagent [14C]-N-ethylmaleimide in a bumetanide-protectable fashion. The specificity of this labeling, and our previous evidence for the existence of an essential sulfhydryl group closely associated with the bumetanide-binding site on the parotid Na(+)-K(+)-Cl-cotransporter (J. Membrane Biol. 112:51-58, 1989), 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 pI of this deglycosylated protein is approximately 6.4. The bumetanide-binding protein was purified using two preparative electrophoresis 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 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 [14C]-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 in this starting extract, this enrichment indicates a high degree of purity of this protein in the 135 kD fraction.
Reshkin
S J
SJ
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, Bethesda, Maryland 20892.
Lee
S I
SI
George
J N
JN
Turner
R J
RJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002034:Bumetanide / Q000737:chemistry*; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D006026:Glycoside Hydrolases / Q000494:pharmacology; D007525:Isoelectric Focusing; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D008970:Molecular Weight; D010306:Parotid Gland / Q000737:chemistry*; D011485:Protein Binding; D011817:Rabbits; D028021:Sodium-Potassium-Chloride Symporters
D000596:Amino Acids; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D006026:Glycoside Hydrolases