Role of phospholipids in the binding of bumetanide to the rabbit parotid Na/K/Cl cotransporter.
It was recently reported (Turner, R.J., George, J.N., 1990, J. Membrane Biol. 113:203-210) that the high affinity bumetanide binding site of the rabbit parotid Na/K/Cl cotransporter could be extracted from a basolateral membrane preparation from this gland using relatively low concentrations of the non-ionic detergent Triton X-100. At the detergent:protein ratios required for complete membrane solubilization bumetanide binding activity in this extract was lost but could be recovered by the addition of crude soybean lipids. In the present paper the ability of various purified lipids to restore high affinity bumetanide binding activity in detergent solubilized rabbit parotid basolateral membranes is studied. We show that the effect of exogenous lipid on the detergent-inactivated bumetanide binding site is to increase the affinity of binding without affecting the number of binding sites. Of the 11 lipid species tested, several relatively minor, negatively charged membrane phospholipids are the most effective in restoring binding activity (phosphatidylserine approximately phosphatidylglycerol greater than phosphatidylinositol greater than cardiolipin), while the major mammalian plasma membrane lipid components phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol are without effect. In addition, we show that in the presence of these minor lipids the affinity of bumetanide binding is considerably increased over that observed in the native membrane (e.g., Kd approximately 0.06 microM in membranes extracted with 0.3% Triton and treated with 0.15% wt/vol phosphatidylserine, vs. Kd approximately 3 microM in native basolateral membranes). This dramatic dependence of bumetanide binding affinity on the presence of certain lipid species suggests that the properties of the bumetanide binding protein in situ may be quite dependent on the minor lipid content of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Corcelli
A
A
Istituto di Fisiologia Generale, Universita degli Studi di Bari, Italy.
Turner
R J
RJ
D000818:Animals; D001665:Binding Sites; D002034:Bumetanide / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D010306:Parotid Gland / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters
D002352:Carrier Proteins; D002712:Chlorides; D010743:Phospholipids; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D012964:Sodium; D011188:Potassium