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

Abstract

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.

• Authors: M Haas, P B Dunham, B Forbush 3rd
• Journal: The American journal of physiology (Am J Physiol) Vol. 260 Issue 4 Pt 1 Pg. C791-804 (Apr 1991) ISSN: 0002-9513 [Print] United States
• PMID: 2018111 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Affinity Labels
• Benzophenones
• Carrier Proteins
• Membrane Proteins
• Sodium-Potassium-Chloride Symporters
• Sulfanilamides
• Bumetanide
• Tritium
• 4-benzoyl-5-sulfamoyl-3-(3-thenyloxy)benzoic acid

Topics

• Affinity Labels
• Animals
• Benzophenones (metabolism)
• Bumetanide (metabolism)
• Carcinoma, Ehrlich Tumor (metabolism)
• Carrier Proteins (isolation & purification, metabolism)
• Cell Fractionation
• Cell Membrane (metabolism, ultrastructure)
• Centrifugation, Density Gradient
• Female
• Kidney (metabolism)
• Kidney Cortex (metabolism)
• Kidney Medulla (metabolism)
• Kinetics
• Membrane Proteins (isolation & purification, metabolism)
• Mice
• Protein Binding
• Sodium-Potassium-Chloride Symporters
• Sulfanilamides (metabolism)
• Tritium