Frequency-domain lifetime fluorometry of double-labeled creatine kinase

Title:

Frequency-domain lifetime fluorometry of double-labeled creatine kinase

Creator:

Gregor, M., Martin Kubala, Evžen Amler, and Jiří Mejsnar

Identifier:

https://cdk.lib.cas.cz/client/handle/uuid:e5d2df33-9cf8-4ffe-914b-240e5746e0ab
uuid:e5d2df33-9cf8-4ffe-914b-240e5746e0ab
issn:0862-8408

Subject:

Fyziologie člověka a srovnávací fyziologie, fyziologie, physiology, creatine kinase molecule, conformational change, FRET, frequency-domain lifetime fluorometry, 14, and 612

Type:

article, články, model:article, and TEXT

Format:

print, bez média, and svazek

Description:

Myofibril-bound creatine kinase EC 2.7.3.2 (CK), a key enzyme of muscle energy metabolism, has been selected for studies of conformational changes that underlie the cellular control of enzyme activity. For fluorescence spectroscopy measurements, the CK molecule was double-labeled with IAF (5-iodoacetamidofluorescein) and ErITC (erythrosin 5'-isothiocyanate). Measurement of fluorescence resonance energy transfer (FRET) from fluorescein to erythrosin was used to obtain information about the donor-acceptor pair distance. Frequency-domain lifetime measurements evaluate the donor-acceptor distance in the native CK molecule as 7.8 nm. The Förster radius equals 5.3 nm with the resolution range from 0.2 to 1.0 nm. Erythrosin-fluorescein labeling (EFL) was tested for artificial conformational changes of the CK molecule with high-salt concentration treatment. The transition distance, defined by His-97 and Cys-283 and derived from a 3D model equals 0.766 nm for the open (inactive) form and 0.277 nm for the closed (reactive) form of the CK molecule. In this way, the resolution range of the used spectroscopy method is significant, concerning the difference of 0.489 nm. Nevertheless, the CK enzyme activity, assessed by the hexokinase-coupled assay, was diminished down to 1 % of the activity of the native enzyme. EFL is suitable for description of conformational behavior implied from the regulation of creatine kinase. However, the observed inhibition restricts EFL to studies of conformational changes during natural catalytic activity., M. Gregor, M. Kubala, E. Amler, J. Mejsnar., and Obsahuje bibliografii

Language:

English

Rights:

http://creativecommons.org/licenses/by-nc-sa/4.0/
policy:public

Source:

Physiological research | 2003 Volume:52 | Number:5

Harvested from:

CDK

Metadata only:

false