Rotation motion and flexibility of calcium(2+),magnesium(2+)-dependent adenosine 5'-triphosphatase in sarcoplasmic reticulum membranes

Abstract

Ca2+,Mg2+-dependent adenosine 5'-triphosphatase (ATPase) in sarcoplasmic reticulum vesicles is labeled with the triplet probe, 5-iodoacetamidoesin. Rotational mobility of the ATPase is investigated by measuring flash-induced transient dichroism of the eosin probe. The absorption anisotropy measured 20 mus after the exciting flash is found to be small at 37 degrees C but increases considerably with decreasing temperature and upon fixation with glutaraldehyde. A purified Ca2+,Mg2+-dependent ATPase preparation partially depleted of membrane lipids exhibits similar properties. The low value of the anisotropy at 37 degrees C is due to the existence of a fast motion which in part is assigned to independent segmental motion of the protein. This internal flexibility of the ATPase may have considerable significance for the functional properties of the enzyme. At times longer than 20 mus, the anisotropy decays with a time constant which varies from approximately 90 mus at 0 degrees C to approximately 40 mus at 37 degrees C. This decay is assigned to rotation of the ATPase about an axis normal to the plane of the membrane. There is some evidence for self-aggregation of the protein at lower temperatures.

Mechanism of Ca2+Transport by Sarcoplasmic Reticulum

“…These motions are much faster than the motions required to account for the enzymatic activity, and it is not known to what extent the motion of the probe reflects the segmental motion of the polypeptide chain to which it is attached. The Arrhenius plot for the rotational mobility of the triplet probe (59,214) indicates two discontinuities at 15°C and at 35°C. Only the discontinuity at 15°C is accompanied by a change in ATPase activity.…”

“…The transition in the temperature dependence of enzyme activity is associated with a change in the rotational motion of the ATPase protein, suggesting either a change in protein conformation or a change in proteinprotein interaction around 15°C-20°C (14,59,212,214,288,289,591). Whether this change is an intrinsic response of the protein to temperature or whether it is triggered by changes in the microviscosity of the environment remains unresolved.…”

“…The SR membranes are dynamic structures in which the phospholipids (513,524) and the Ca 2+-transport ATPase molecules (14,59,212,214,288,289,363,591) move rapidly.…”

Long-lifetime lipid probe containing a luminescent metal-ligand complex

“…[5][6][7] However, the use of phosphorescence requires the complete exclusion of oxygen, and the phosphorescence time-zero anisotCell membranes are predominantly composed of ropies are often low. phospholipids, which are spectroscopically silent In this communication we describe an alternain the ultraviolet and visible regions of the spective method to obtain luminescent probes with trum.…”

Fluorescence Studies on N-(3-Pyrene)Maleinimide-Labeled Sarcoplasmic Reticulum ATPase in Native and Solubilized Membranes

“…1 pyrenem aleinim ide m ol ecule per 100 ATPase molecules), suggests an ad ditional motion of pyrenem aleinim ide which does not monitor the rotation of the entire ATPase m ol ecule within the membrane. This kind o f segmental motion was originally described by Bürkli and Cherry [23] and Restall et al [24], using flashinduced dichroism measurements o f the triplet probe 5-iodoacetamideosin bound to sarcoplasm ic sarcoplasmic reticulum ATPase. The occurrence of segmental motion was also supported by the fact that nonsolubilized vesicles showed a small decrease of the pyrenemaleinimide polarization at increasing temperature.…”