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Proceedings Paper

Measurement of conformational states of Ca2+-ATPase in sarcoplasmic reticulum using phosphorescence anisotropy
Author(s): Liqun Yang; A. N. Rubtsov; Daniel McStay; A. A. Boldyrev; Peter J. Quinn
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Paper Abstract

Ca2+-activated adenosine triphosphatase isolated from rabbit muscle sarcoplasmic reticulum has been specifically labelled at a single lysine located at the putative ATP binding site with the triplet probe, eosin-5'-isothiocyanate. Labelled microsomes were suspended in buffer containing different cations to shift the enzyme to one or the other of its conformeric states, denoted E1 and E2. Samples in the different conformations were excited with a short laser pulse from a frequency doubled Nd:YAG laser. The time-resolved phosphorescence of the protein-bound probe was measured using a microcomputer-based dual- channel phosphorimeter over a temperature span of 2 degree(s) - 42 degree(s) C, and the emission anisotropy computed. The study suggests that in both conformeric states, the enzyme consists of different protein aggregates, however, the relative population of different protein aggregates may be different with the E2 form of the ATPase tending to form aggregates of larger size and the E1 form of the enzyme tending to form smaller aggregates. A more precise estimation of the sizes of the protein rotating species depends on accurate determination of the orientation of the label on the protein molecules.

Paper Details

Date Published: 1 February 1994
PDF: 8 pages
Proc. SPIE 2083, Microscopy, Holography, and Interferometry in Biomedicine, (1 February 1994); doi: 10.1117/12.167417
Show Author Affiliations
Liqun Yang, The Robert Gordon Univ. (United Kingdom)
A. N. Rubtsov, Moscow State Univ. (Russia)
Daniel McStay, The Robert Gordon Univ. (United Kingdom)
A. A. Boldyrev, Moscow State Univ. (Russia)
Peter J. Quinn, King's College London (United Kingdom)


Published in SPIE Proceedings Vol. 2083:
Microscopy, Holography, and Interferometry in Biomedicine
Adolf Friedrich Fercher; Aaron Lewis; Halina Podbielska; Herbert Schneckenburger; Tony Wilson, Editor(s)

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