[H-3] BUMETANIDE BINDING TO MOUSE KIDNEY MEMBRANES - IDENTIFICATION OF CORRESPONDING MEMBRANE-PROTEINS

Crude plasma membranes from whole mouse kidneys have two classes of [H-3]-bumetanide binding sites. High-affinity sites (K1/2 congruent-to 0.04-mu-M; B(max) = 1-2 pmol/mg protein) are similar to those identified on dog kidney membranes (B. Forbush and H. C. Palfrey. J. Biol. Chem. 258: 11787-11792, 1983) both with respect to affinity and in that Na, K, and Cl are required for [H-3]bumetanide binding. Low-affinity sites (K1/2 congruent-to 1-mu-M; B(max) = 7-14 pmol/mg) are unaffected by removal of these ions; such sites are not seen with dog kidney. When mouse kidney membranes are photolabeled with 4-[H-3]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid ([H-3]BSTBA), a photoreactive bumetanide analogue, specific incorporation of the label is seen in two regions. As with dog kidney [M. Haas and B. Forbush. Am. J. Physiol. 253 (Cell Physiol. 22): C243-C252, 1987], an approximately 150-kDa protein is labeled with high affinity (K1/2 congruent-to 0.05-mu-M). This labeling also requires Na, K, and Cl and appears to correspond to the high affinity [H-3]bumetanide binding sites and to the Na-K-Cl co-transport system. A second peak of [H-3]BSTBA photolabeling, centered at approximately 75 kDa, incorporates the label with lower affinity (K1/2 = 2-3-mu-M). The photolabeling at approximately 75 kDa is unaffected by Na, K, and Cl concentrations and thus may correspond, at least in part, to the low-affinity [H-3]bumetanide binding sites. Western blot analysis of [H-3]BSTBA-labeled mouse kidney membranes was performed using an antiserum raised to proteins of approximately 82 and approximately 39 kDa isolated from mouse Ehrlich ascites tumor cells using a bumetanide affinity gel (P. B. Dunham, F. Jessen, and E. K. Hoffmann. Proc. Natl. Acad. Sci. USA 87: 6828-6832, 1990). This antiserum cross-reacts with a approximately 150-kDa mouse kidney protein, the staining profile of which on Western blot corresponds very closely to the peak of specific [H-3]BSTBA incorporation in this region. The antiserum also reacts with proteins in the range of 65-85 kDa, overlapping the low-affinity peak of [H-3]BSTBA incorporation.