Preparation of Na,K-ATPase specifically modified on the anti-fluorescein antibody-inaccessible site by fluorescein 5'-isothiocyanate.
Specific labeling is required for energy transfer measurements and to avoid artifacts in the use of fluorophores as reporter groups. Therefore, a method for specific modification by one of the most popular reagents for P-type ATPases (fluorescein 5'-isothiocyanate) has been developed. Sulfhydryl reagents protected against modification of cysteine residues, and treatment with dithiothreitol eliminated a slow doubling of the fluorescence of conventionally modified Na,K-ATPase upon dilution that is attributed to disappearance of self-energy transfer. Removal of nonspecifically bound fluorescein was also confirmed by titration of the modified Na, K-ATPase with anti-fluorescein antibody and by time resolution of the fluorescence change when the modified enzyme was mixed with Na(+) in a stopped-flow instrument. The only fluorescence change when specifically modified Na,K-ATPase was mixed with Na(+) was the signal from fluorescein at the antibody-inaccessible, substrate-protectable site that reports the conformational change in unphosphorylated enzyme. The magnitude of the fluorescence change reporting the conformational change increased from between 8 and 12% to between 25 and 30% without affecting the kinetic constants estimated from titrations with Na(+) and K(+). The method should be generally applicable to the preparation of specifically labeled P-type pumps for use in kinetic and equilibrium titrations or energy transfer measurements.
Lin
S H
SH
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch at Galveston, 77555-1055, USA.
Faller
L D
LD
D000818:Animals; D001666:Binding Sites, Antibody; D004735:Energy Transfer; D019793:Fluorescein / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013552:Swine
D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D019793:Fluorescein