AJP - Cell Physiology, Vol 260, Issue 4 C791-C804, Copyright © 1991 by American Physiological Society

ARTICLES

[3H]bumetanide binding to mouse kidney membranes: identification of corresponding membrane proteins

M. Haas, P. B. Dunham and B. Forbush 3rd
Department of Pathology, University of Chicago, Illinois 60637.

Crude plasma membranes from whole mouse kidneys have two classes of [3H]bumetanide binding sites. High-affinity sites (K1/2 approximately equal to 0.04 microM; Bmax = 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 [3H]bumetanide binding. Low-affinity sites (K1/2 approximately equal to 1 microM; Bmax = 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-[3H]benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid [( 3H]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 approximately equal to 0.05 microM). This labeling also requires Na, K, and Cl and appears to correspond to the high-affinity [3H]bumetanide binding sites and to the Na-K-Cl cotransport system. A second peak of [3H]BSTBA photolabeling, centered at approximately 75 kDa, incorporates the label with lower affinity (K1/2 = 2-3 microM). 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 [3H]bumetanide binding sites. Western blot analysis of [3H]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 [3H]BSTBA incorporation in this region. The antiserum also reacts with proteins in the range of 65-85 kDa, overlapping the low-affinity peak of [3H]BSTBA incorporation.

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