Interaction of the fluorescent probe RH421 with ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments.
Fluorescence titrations have shown that the voltage-sensitive probe RH421 interacts with the water-soluble protein ribulose-1,5-bisphosphate carboxylase/oxygenase and with Na+,K(+)-ATPase membrane fragments. The probe exhibits significantly different fluorescence excitation spectra in pure lipid and pure protein environments. Experiments with a range of polyamino acids showed interactions of the probe with tyrosine, lysine and arginine residues. At saturating RH421 concentrations (> or = microM) the probe quenches 60-75% of the total tryptophan fluorescence of the Na+,K(+)-ATPase preparation. Inhibition of the hydrolytic activity of the Na+,K(+)-ATPase occurs at RH421 concentrations in the micromolar range. This may be due to a probe-induced change in membrane fluidity. The sensitivity of the probe towards conformational changes of the Na+,K(+)-ATPase decreases hyperbolically as one increases the probe concentration. The decrease in sensitivity correlates well with association of the probe in the vicinity of membrane protein, as measured by tryptophan quenching. These results have important practical consequences for the application of RH421 as a voltage indicator in membrane preparations. Based on these and previously reported results, the fluorescent response of RH421 to the ATP-induced conformational change of the Na+,K+-ATPase is consistent with either a redistribution of dye from the liquid-crystalline lipid matrix into the vicinity of membrane protein or a reorganisation of the lipids surrounding the protein into a more rigid structure caused by the conformational change of the protein.
Frank
J
J
Department of Physical Chemistry, Fritz-Haber-Institut der Max-Planck-Gesellschaft, Berlin, Germany.
Zouni
A
A
van Hoek
A
A
Visser
A J
AJ
Clarke
R J
RJ
D002462:Cell Membrane / Q000201:enzymology; D005456:Fluorescent Dyes; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000494:pharmacology; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013343:Styrenes / Q000494:pharmacology
D005456:Fluorescent Dyes; D010446:Peptide Fragments; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D012273:Ribulose-Bisphosphate Carboxylase; D000254:Sodium-Potassium-Exchanging ATPase