Preparation of Na,K-ATPase Specifically Modified on the Anti-fluorescein Antibody-Inaccessible Site by Fluorescein 5′-Isothiocyanate
| Autor: | Larry D. Faller, Shwu-Hwa Lin |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Conformational change
Swine Biophysics Kidney Biochemistry Dithiothreitol chemistry.chemical_compound Animals Fluorescein Na+/K+-ATPase Molecular Biology Chromatography Chemistry Cell Biology Fluorescence Kinetics Spectrometry Fluorescence Energy Transfer Reagent Titration Binding Sites Antibody Sodium-Potassium-Exchanging ATPase Fluorescein-5-isothiocyanate Cysteine |
| Zdroj: | Analytical Biochemistry. 287:303-312 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.2000.4828 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect