Purification of proteins of the Na/Cl cotransporter from membranes of Ehrlich ascites cells using a bumetanide-sepharose affinity column

Abstract

Bumetanide-binding proteins were isolated from membranes of Ehrlich ascites tumor cells by affinity chromatography. An affinity column was constructed with the active moiety of bumetanide as a ligand using 4'-azidobumetanide, a photoactive analogue which inhibits Na/Cl cotransport in Ehrlich cells with high specificity. Covalent binding of the 4'-azidobumetanide with Sepharose was promoted by photolysis. Membranes isolated from Ehrlich cells were solubilized with n-octylglucoside. Solubilized proteins retarded by the affinity column were readily eluted by bumetanide. In reducing gels the major proteins eluted by bumetanide were approximately 76 kDa and 38-39 kDa. There were also two proteins of 32 to 35 kDa eluted in lesser amounts. No proteins retarded by the affinity column were eluted with extensive washing without bumetanide. Furthermore, bumetanide eluted no proteins from a "control" column lacking the specific ligand. Upon rechromatography with bumetanide in solution, bumetanide-eluted proteins were not retarded, but their purity was increased by the retardation of contaminating proteins. Bumetanide-binding protein purified in this manner were characterized further by electrophoresis in nonreducing, nondenaturing gels.

Mechanisms in Volume Regulation in Ehrlich Ascites Tumor Cells

“…The inhibition of the furosemide-sensitive cotransport system in Ehrlich cells by these drugs is, however, not mediated by cAMP or cGMP [Geek and Pfeiffer, 1985]. As mentioned previously (see section 6) bumetanide binding studies indi cate that the activation of the cotransport system in Ehrlich cells during RVI does not involve the recruitment of new transport sites [Feit et al, 1988).…”

“…The bumetanide-sensitive cotransport system in Ehrlich cells is quiescent under isotonic steady-state conditions , Nevertheless, resting cells showed the same number of bumetanidebinding sites as the cells where the cotrans port system was activated by cell shrinkage, indicating that recruitment of new transport sites is not involved in the activation of the cotransport system during the RVI response in these cells [Feit et al, 1988],…”

Solubilization and partial purification of the rabbit parotid Na/K/Cl-dependent bumetanide binding site

“…In many of these studies covalent modification (inactivation) of the transporter precludes a direct measurement of its functional properties [5,13,14,17]. In the remainder [7,28], the function of the final purified protein fraction has not yet been demonstrated.…”

“…More recently, Pewitt et al [28] have demonstrated that, when solubilized calf renal outer medullary membranes are passed over, an affinity chromatography column consisting of 4-paminobumetanide coupled to Affigel-10, a 160-kDa protein is retained and can be subsequently eluted with excess bumetanide. Feit et al [7] have also used affinity chromatographic techniques with a bumetanide analogue to identify proteins with molecular weights -76 and -38 kDa in membranes from Ehrlich ascites tumor cells. At present it is not clear whether the above lack of consensus regarding the molecular weight of the Na/K/CI cotransporter is due to the identification of various subunits or degradation products of the protein, to differences in the cotransporter between tissues, or to methodological difficulties.…”

Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters

“…After the discovery of electrically silent cotransport mechanisms in mammalian cells and tissues 25 years ago (138,340), several laboratories undertook unrewarded attempts to identify the proteins responsible for such a transport system (94,107,121,197,251,406). However, the major breakthrough in this field came in the early 1990s from two different laboratories that were able to identify the genes responsible for Na ϩ -Cl Ϫ (136, 137) and Na ϩ -K ϩ -2Cl Ϫ cotransporters (136,311,440).…”