Phosphorescence depolarization and the rotation mobility of Na+,       K+-activated ATPase in crude microsomal membranes

Liqun Yang; Olga Lopina; Daniel McStay; Alan J. Rogers; Peter J. Quinn

doi:10.1117/12.60697

1 February 1993 Phosphorescence depolarization and the rotation mobility of Na⁺, K⁺-activated ATPase in crude microsomal membranes

Liqun Yang, Olga Lopina, Daniel McStay, Alan J. Rogers, Peter J. Quinn

Author Affiliations +

Optical Engineering, Vol. 32, Issue 2, (February 1993). https://doi.org/10.1117/12.60697

Abstract

The Na⁺, K⁺-adenosine triphosphatase (ATPase) in microsomal membrane vesicles is covalently labeled with the triplet probe eosin 5'-isothiocyanate. Rotational mobility of the protein is investigated by measuring the time-resolved depolarization of the emitted phosphorescence from the triplet state of eosin, induced by a laser pulse. The probe is attached either nonspecifically to the protein or under conditions where the eosin is attached specifically to a lysine residue located atthe putative ATP binding site. The total anisotropy of the emission is found to be almost constant when measured over the temperature range 10 to 25°C. The anisotropy value is relatively small when the label is bound nonspecifically to the binding sites of the protein, but markedly increases when specifically bound to the protein, suggesting that the independent motion of the probe is constrained at this site. The anisotropy decay curve obtained from the specifically labeled protein shows a clearly biphasic character, and is composed of a rapidly rotating component with a rotational correlation time of 20 to 5 μs and a slower rotating component with a rotational correlation time of 250 to 90 μs in the temperature range 10 to 25°C. These motions are individually assigned to the segmental motion of the polypeptide chain and rotation of the whole protein about its axis normal to the plane of the membrane, respectively. It is estimated that about 80% ofthe total anisotropy signal is contributed by the fast rotation, and the remainder results from slow rotation.

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Liqun Yang, Olga Lopina, Daniel McStay, Alan J. Rogers, and Peter J. Quinn "Phosphorescence depolarization and the rotation mobility of Na⁺, K⁺-activated ATPase in crude microsomal membranes," Optical Engineering 32(2), (1 February 1993). https://doi.org/10.1117/12.60697

Published: 1 February 1993

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