[3H]bumetanide binding to mouse kidney membranes: identification of corresponding membrane proteins.
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.
Haas
M
M
Department of Pathology, University of Chicago, Illinois 60637.
Dunham
P B
PB
Forbush
B
B
D000345:Affinity Labels; D000818:Animals; D001577:Benzophenones / Q000378:metabolism; D002034:Bumetanide / Q000378:metabolism*; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D005260:Female; D007668:Kidney / Q000378:metabolism*; D007672:Kidney Cortex / Q000378:metabolism; D007679:Kidney Medulla / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D028021:Sodium-Potassium-Chloride Symporters; D013424:Sulfanilamides / Q000378:metabolism; D014316:Tritium
D000345:Affinity Labels; D001577:Benzophenones; D002352:Carrier Proteins; D008565:Membrane Proteins; D028021:Sodium-Potassium-Chloride Symporters; D013424:Sulfanilamides; D002034:Bumetanide; D014316:Tritium; C055204:4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid