Preparation of Na,K-ATPase Specifically Modified on the Anti-fluorescein Antibody-Inaccessible Site by Fluorescein 5′-Isothiocyanate
✍ Scribed by Shwu-Hwa Lin; Larry D. Faller
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 114 KB
- Volume
- 287
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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.