Spectroscopic analysis of halothane binding to the plasma membrane Ca2+-ATPase.
The intrinsic tryptophan (Trp) fluorescence of the plasma membrane Ca2+-ATPase (PMCA) is significantly quenched by halothane, a volatile anesthetic common in clinical practice. It has been proposed that halothane inhibition of the Ca2+-ATPase activity results from conformational changes following anesthetic binding in the enzyme. We have investigated whether the observed quenching reflects halothane binding to PMCA. We have shown that the quenching is dose dependent and saturable and can be fitted to a binding curve with an equilibrium constant K(Hal) = 2.1 mM, a concentration at which the anesthetic approximately half-maximally inhibits the Ca2+-ATPase activity. The relatively low sensitivity of halothane quenching of Trp fluorescence to the concentration of phosphatidylcholine and detergent in the PMCA preparation concurs with the quenching resulting from anesthetic binding in the PMCA molecule. Analysis of the Trp fluorescence quenching by acrylamide indicates that the Trp residues are not considerably exposed to the solvent (Stern-Volmer quenching constant of 2.9 M(-1)) and do not differ significantly in their accessibility to halothane. Other volatile anesthetics, diethyl ether and diisopropyl ether, reduce the quenching caused by halothane in a dose-dependent manner, suggesting halothane displacement from its binding site(s). These observations indicate that halothane quenching of intrinsic Trp fluorescence of PMCA results from anesthetic binding to the protein. The analysis, used as a complementary approach, provides new information to the still rudimentary understanding of the process of anesthetic interaction with membrane proteins.
Lopez
M M
MM
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409, USA. qemar@ttacs.ttu.edu
Kosk-Kosicka
D
D
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D018685:Anesthetics, Inhalation / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D004987:Ethers / Q000494:pharmacology; D006221:Halothane / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D010713:Phosphatidylcholines; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan; D014508:Urea / Q000494:pharmacology
D000178:Acrylamides; D018685:Anesthetics, Inhalation; D004987:Ethers; D010713:Phosphatidylcholines; D020106:Acrylamide; D014364:Tryptophan; D014508:Urea; D000252:Calcium-Transporting ATPases; D006221:Halothane