Rotational diffusion of calcium-dependent adenosine-5'-triphosphatase in sarcoplasmic reticulum: a detailed study.
The Ca2+-Mg2+ adenosine-5'-triphosphatase (ATPase) in sarcoplasmic reticulum has been covalently labeled with the phosphorescent triplet probe erythrosinyl 5-isothiocyanate. The rotational diffusion of the protein in the membrane at 25 degrees C was examined by measuring the time dependence of the phosphorescence emission anisotropy. Detailed analysis of both the total emission S(t) = Iv(t) + 2IH(t) and anisotropy R(t) = [Iv(t) - IH(t)]/[Iv(t) + 2IH(t)] curves shows the presence of multiple components. The latter is incompatible with a simple model of protein movement. The experimental data are consistent with a model in which the sum of four exponential components defines the phosphorescence decay. The anisotropy decay corresponds to a model in which the phosphor itself or a small phosphor-bearing segment reorients on a sub-microsecond time scale about an axis attached to a larger segment, which in turn reorients on a time scale of a few microseconds about an axis fixed in the frame of the ATPase. A fraction of the protein molecules rotate on a time scale of 100-200 microseconds about the normal to the bilayer, while the rest are rotationally stationary, at least on a sub-millisecond time scale.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D004923:Erythrosine / Q000031:analogs & derivatives; D005454:Fluorescence Polarization; D017879:Isothiocyanates; D008956:Models, Chemical; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D017879:Isothiocyanates; C022232:erythrosine isothiocyanate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D004923:Erythrosine