Measurement of conformational states of Ca2+-ATPase in sarcoplasmic reticulum using phosphorescence anisotropy

Liqun Yang; A. N. Rubtsov; Daniel McStay; A. A. Boldyrev; Peter J. Quinn

doi:10.1117/12.167417

1 February 1994 Measurement of conformational states of Ca²⁺-ATPase in sarcoplasmic reticulum using phosphorescence anisotropy

Liqun Yang, A. N. Rubtsov, Daniel McStay, A. A. Boldyrev, Peter J. Quinn

Author Affiliations +

Proceedings Volume 2083, Microscopy, Holography, and Interferometry in Biomedicine; (1994) https://doi.org/10.1117/12.167417
Event: Europto Biomedical Optics '93, 1993, Budapest, Hungary

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.

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Liqun Yang, A. N. Rubtsov, Daniel McStay, A. A. Boldyrev, and Peter J. Quinn "Measurement of conformational states of Ca²⁺-ATPase in sarcoplasmic reticulum using phosphorescence anisotropy", Proc. SPIE 2083, Microscopy, Holography, and Interferometry in Biomedicine, (1 February 1994); https://doi.org/10.1117/12.167417

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KEYWORDS

Proteins

Anisotropy

Phosphorescence

Temperature metrology

Molecules

Acquisition tracking and pointing

Calcium

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