Phosphorescence depolarization and measurement in the rotation of eosin-labeled Na+ K+-activated ATPase in microsomal membranes

摘要

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