Changes in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ ATPase enzyme in the presence of terbium: a time-resolved x-ray diffraction study

Abstract

The design of the time-resolved x-ray diffraction experiments reported in this and an accompanying paper was based on direct measurements of enzyme phosphorylation using [gamma-32P]ATP that were employed to determine the extent to which the lanthanides La3+ and Tb3+ activate phosphorylation of the Ca2+ATPase and their effect on the kinetics of phosphoenzyme formation and decay. We found that, under the conditions of our experiments, the two lanthanides are capable of activating phosphorylation of the ATPase, resulting in substantial levels of phosphoenzyme formation and they slow the formation and dramatically extend the lifetime of the phosphorylated enzyme conformation, as compared with calcium activation. The results from the time-resolved, nonresonance x-ray diffraction work reported in this paper are consistent with the enzyme phosphorylation experiments; they indicate that the changes in the profile structure of the SR membrane induced by terbium-activated phosphorylation of the ATPase enzyme are persistent over the much longer lifetime of the phosphorylated enzyme and are qualitatively similar to the changes induced by calcium-activated phosphorylation, but smaller in magnitude. These results made possible the time-resolved, resonance x-ray diffraction studies reported in an accompanying paper utilizing the resonance x-ray scattering from terbium, replacing calcium, to determine not only the location of high-affinity metal-binding sites in the SR membrane profile, but also the redistribution of metal density among those sites upon phosphorylation of the Ca2+ATPase protein, as facilitated by the greatly extended lifetime of the phosphoenzyme.

Changes in the relative occupancy of metal-binding sites in the profile structure of the sarcoplasmic reticulum membrane induced by phosphorylation of the Ca2+ATPase enzyme in the presence of terbium: a time-resolved, resonance x-ray diffraction study

“…The methods and procedures used for the preparation of the hydrated, oriented multilayer specimens of isolated SR membranes, the collection of their lamellar x-ray diffraction data, and initial diffraction data reduction are described in an accompanying paper (Asturias et al, 1994), and will not be presented here. The result of the first phase of data reduction were sets of three background scattering-corrected, lamellar intensity functions I(Z*, Eedge), and I(z*, Eedge+a) per stable specimen, where Eedge represents the x-ray energy corresponding to the terbium LIM absorption edge (7506 eV), and A = +100 eV.…”

“…The result of the first phase of data reduction were sets of three background scattering-corrected, lamellar intensity functions I(Z*, Eedge), and I(z*, Eedge+a) per stable specimen, where Eedge represents the x-ray energy corresponding to the terbium LIM absorption edge (7506 eV), and A = +100 eV. For the time-resolved, nonresonance x-ray diffraction study, only the edge-energy lamellar intensity functions I,(z*, Eedge) were analyzed further to derive the relative electron density profile for the multilayer unit cell containing two apposed profiles of the SR membrane, and the nonresonant changes in the unit cell and single SR membrane profile upon flash photolysis of caged ATP and subsequent protein phosphorylation in the multilayer specimens were determined (Asturias et al, 1994).…”

“…Significant structural changes were found upon Th3+-activated phosphorylation of the Ca2+ATPase, and they were similar in nature, but smaller in magnitude, to those found upon Ca2+-activated enzyme phosphorylation. The results from these biochemical/kinetic studies and time-resolved, nonresonance x-ray diffraction studies are described in an accompanying paper (Asturias et al, 1994).…”

Toxicological and cytophysiological aspects of lanthanides action.

“…It is suggested that Dy 3+ inhibits this enzyme much more than other lanthanide ions (Van der Laarse et al, 1995). La 3+ and Tb 3+ trigger phosphorylation of Ca 2+ -ATPase and increase the half-life of the phosphorylated enzyme (Asturias et al, 1994). Lanthanum ions can inhibit Mg 2+ -ATPases and choline esterases and activate kinase C (Wadkins et al, 1998).…”

The structure of the Ca2+-ATPase of sarcoplasmic reticulum.

“…This may be explained by assuming that the E 1~P form of the enzyme is the dominant intermediate during the steady state. La 3+ and Tb 3+ also activate the phosphorylation of Ca 2+ -ATPase (Asturias et al, 1994b) and the Tb 3+ induced phosphoenzyme formation was accompanied by decrease in electron density in the outer phospholipid monolayer and an increase in the inner phospholipid monolayer. These changes are qualitatively similar to those observed in the presence of Ca 2+ (Herbette et al, 1985;Blasie et al, 1990) but smaller in magnitude due perhaps to the lower steady state concentrations of E~P found in the presence of Tb 3+ (Asturias et al, 1994b).…”