Mechanism of modulation of the plasma membrane Ca(2+)-ATPase by arachidonic acid.

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.

Effects of gamma-irradiation on the membrane ATPases of human erythrocytes from transfusional blood concentrates.

Irradiation of blood derivatives is employed in blood banks to avoid transfusion-associated graft-vs-host disease. As irradiation can damage membranes and membrane proteins by generation of reactive oxygen species, we investigated whether the membrane permeability, Na(+),K(+)-ATPase, and Ca(2+)-ATPase from red blood cell plasma membranes were altered by gamma-irradiation. Whole blood was collected from healthy donors and concentrated to 90% cell fraction. Within 24 h of collection, blood concentrates were irradiated with 25 Gy of gamma-radiation. At days 1, 7, 14, and 28 post-irradiation, fractions were removed and centrifuged. Na(+),K(+)-ATPase and Ca(2+)-ATPase activities from ghost membranes were assessed by gamma-(32)P-ATP hydrolysis. The Na(+),K(+)-ATPase was not immediately affected by irradiation, but it was inhibited by 40% by day 14 and until day 28. The Ca(2+)-ATPase was unaltered by irradiation. The rate and the maximal (45)Ca(2+) uptake from re-sealed inside-out vesicles were reduced, and the passive efflux of (45)Ca(2+) was increased. Thus, irradiation of blood concentrates increased the plasma membrane permeability to monovalent and divalent cations and would change ion homeostasis and cell function. We recommend the use of irradiated blood within a period shorter than 14 days after irradiation.

Inhibition of plasma membrane Ca2+-ATPase by heparin is modulated by potassium.

Heparin is related to several protein receptors that control Ca2+ homeostasis. Here, we studied the effects of heparin on the plasma membrane Ca2+-ATPase from erythrocytes. Both ATP hydrolysis and Ca2+ uptake were inhibited by heparin without modification of the steady-state level of phosphoenzyme formed by ATP. Calmodulin did neither modify the inhibition nor the binding of heparin. Inhibition by heparin was counteracted by K+ but not by Li+. This effect was extended to other sulfated polysaccharides with high number of sulfate residues. Hydrolysis of p-nitrophenylphosphate was equally inhibited by heparin. No evidence for enzyme uncoupling was observed: Ca2+ uptake and ATP hydrolysis remained tightly associated at any level of heparin, and heparin did not increase the passive Ca2+ efflux of inside-out vesicles. Vanadate blocked this efflux, indicating that the main point of Ca2+ escape from these vesicles was linked to the Ca2+ pump. It is discussed that sulfated polysaccharides may physiologically increase the steady-state level of Ca2+ in the cytosol by inhibiting the Ca2+ pumps in a K+ (and tissue) regulated way. It is suggested that heparin regulates the plasma membrane Ca2+-ATPase by binding to the E2 conformer.

Converting troponin C into calmodulin: effects of mutations in the central helix and of changes in temperature.

Calmodulin (CaM) and troponin C (TnC) are the most similar members of EF-hand family and show few differences in the primary structure. Here, we use mutants of troponin that mimic calmodulin and changes in temperature to investigate the factors that determine their specificity as regulatory proteins. Using a double mutant of troponin that resembles calmodulin in lacking both the N-terminal helix and KGK(91-93) we observe a small difference from troponin in binding to the erythrocyte Ca(2+)-ATPase, and an improvement in enzyme activation. A triple mutant, where in addition, the residues 88-90 are replaced with the corresponding sequence from calmodulin is equivalent to calmodulin in maximal activation, and it restores protein ability to increase Ca(2+) affinity for the enzyme. However, this mutant also binds less tightly (1/100) than calmodulin. Remarkably, a decrease in temperature has a more marked effect in protein binding than either mutation, reducing the difference in affinities to 18-fold, but without any improvement in their ability to increase Ca(2+) affinity for the enzyme. Spectroscopic analysis of hydrophobic domain exposure in EF-hand proteins was carried out using 8-anilino-1-naphthalenesulfonic acid (ANS). The probe shows a much higher fluorescence when bound to the complex Ca(4)-calmodulin than to Ca(4)-troponin. Decreasing the temperature exposes additional hydrophobic regions of troponin. Changing the Mg(2+) concentration does not affect their bindings to the enzyme. It is suggested that the requirements for troponin to mimic calmodulin in binding to the target enzyme, and those for activating it, are met by different regions of the protein.