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Identification, characterization and purification of a 160 kD bumetanide-binding glycoprotein from the rabbit parotid
S J Reshkin1, S I Lee, J N George
1Clinical Investigations and Patient Care Branch, National Institute of Dental Research, Bethesda, Maryland 20892.
Abstract:
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.
Insights
Researchers identified a 160 kD protein in rabbit parotid membranes that binds bumetanide, a diuretic. This protein, crucial for bumetanide binding, was purified and characterized after deglycosylation.
Area of Science:
- Membrane protein biochemistry
- Ion transport mechanisms
- Renal physiology
Background:
- The Na(+)-K(+)-Cl-cotransporter is a key protein in ion transport.
- Bumetanide is a diuretic that specifically inhibits this cotransporter.
- Previous studies suggested a sulfhydryl group is essential for bumetanide binding.
Purpose of the Study:
- To identify and characterize the protein responsible for bumetanide binding in rabbit parotid basolateral membranes.
- To investigate the role of N-linked oligosaccharides in the protein's structure and function.
Main Methods:
- Labeling of membrane proteins with [14C]-N-ethylmaleimide in a bumetanide-protectable manner.
- Enzymatic deglycosylation using endoglycosidase F/N-glycosidase F.
- Purification via two-step preparative electrophoresis.
- Analysis of molecular weight and isoelectric point (pI).
Main Results:
- A 160 kD protein was identified, labeled by [14C]-N-ethylmaleimide, and protected by bumetanide.
- Deglycosylation reduced the protein's apparent molecular weight to 135 kD, with a pI of approximately 6.4.
- Purification yielded a 135 kD fraction with approximately 48-fold enrichment of the bumetanide-binding protein.
Conclusions:
- The 160 kD protein, which becomes 135 kD after deglycosylation, represents the parotid bumetanide-binding site.
- This protein is likely a component or the entirety of the bumetanide-binding site on the Na(+)-K(+)-Cl-cotransporter.
- The study successfully purified and characterized a key protein involved in diuretic action.