Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosine triphosphatase as reflected by organic solvent induced perturbation

Champeil, Philippe; Guillain, Florent; Venien, C.; Gingold, M P

doi:10.1021/bi00322a012

Cited by 137 publications

(85 citation statements)

References 45 publications

(84 reference statements)

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“…SR Membrane Preparation-SR vesicles were isolated from rabbit skeletal muscle according to de Meis and Hasselbach (29) and Champeil et al (30). Before the preparation, the rabbits were subjected to a 48-h starvation diet (31).…”

Section: Methodsmentioning

confidence: 99%

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Lenoir

Picard

Møller

et al. 2004

Journal of Biological Chemistry

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Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6 -7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca 2؉ altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca 2؉ is, however, more significantly reduced (20 -30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca 2 E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca 2؉ , highlighting the long range interactions between L6 -7 and the nucleotide-binding site. We propose that these mutations in L6 -7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.Muscle relaxation occurs by of the removal of calcium from the cytosol and its accumulation into sarcoplasmic reticulum (SR) 1 through an active transport that consumes ATP (1, 2). In fast twitch muscle this transport is achieved by the SERCA1a isoform of Ca 2ϩ -ATPase (3), with a stoichiometry of two calcium ions transported per ATP molecule hydrolyzed (see reviews in Refs. 4 and 5). SR Ca 2ϩ -ATPase belongs to the family of P-type ATPases, which transport cations such as H ϩ , Na ϩ , K ϩ , or Ca 2ϩ across the membrane and share a common mechanism that involves autophosphorylation of the protein (see review in Ref. 6): transport is achieved through a reversible cycle, during which the ATPase is thought to sequentially adopt two main conformations (7, 8), E1 and E2, with a high and low affinity for the transported ion, and the overall cycle corresponds to completion of the following scheme: E2 3 Ca 2 E1 3 Ca 2 E1 ϳ P 3 E2P 3 E2 (for review, see Refs. 9 and 10). Much effort has gone into determining the structural organization of SR Ca 2ϩ -ATPase from primary structure predictions (11), electron microscopy (12), and three-dimensional crystallogenesis (13-15) (for a recent review see Ref. 16). Finally, the structure has been solved at the atomic level, first in a Ca 2 E1 state (14) and then in a thapsigargin-stabilized E2 state (15). T...

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Section: Methodsmentioning

confidence: 99%

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Lenoir

Picard

Møller

et al. 2004

Journal of Biological Chemistry

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“…Bell-shaped titration curves were observed previously for the calcium pump when the amount (6,8,29) or rate (7) of E-P formation from P i measured by counting 32 P was plotted against [Mg 2ϩ ]. The decrease in E-P was attributed to a low affinity inhibitory Mg 2ϩ site (8) or Mg 2ϩ binding to Ca 2ϩ sites causing a shift in the conformational equilibrium toward unreactive E 1 (6). However, the dip in the surface formed by the data points in Fig.…”

Section: Discussionmentioning

confidence: 73%

“…The theoretical phosphorylation level for a molecular mass of 109,485 Da is 9.13 nmol mg Ϫ1 . Therefore, 65 Ϯ 6% of the Lowry protein in the membranes was functional enzyme, assuming complete phosphorylation in the organic solvent-water mixture at the lower pH (8).…”

Section: Methodsmentioning

confidence: 99%

“…Most published studies of purified enzyme in aqueous solution support this interpretation (3)(4)(5)(6)(7). However, a study in an organic solventwater mixture concluded that the true substrate is Mg⅐P i (8), and recently the equation for the half-maximum Mg⅐P i concentration was used to interpret an increased half-maximum P i concentration when the upstream threonine in a signature amino acid sequence for P-type pumps (DKTGT) was changed to serine (9). The interpretation given to most studies of Na,KATPase is also that Mg 2ϩ and P i bind randomly (10 -12).…”

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confidence: 99%

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Evidence Calcium Pump Binds Magnesium before Inorganic Phosphate

Nagy

Kane

Tran

et al. 2005

Journal of Biological Chemistry

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Calcium pump-catalyzed18 O exchange between inorganic phosphate and water was studied to test the hypothesis that all P-type pumps bind Mg 2؉ before P i and validate utilization of the rate equation for ordered binding to interpret differences between site-directed mutants and wild-type enzyme. The results were remarkably similar to those obtained earlier with sodium pump (Kasho, V. N., Stengelin, M., Smirnova, I. N., and Faller, L. D. (1997) Biochemistry 36, 8045-8052). The equation for ordered binding of Mg 2؉ before P i fit the data best with only a slight chance (0.6%) of P i binding to apoenzyme. Therefore, P i is the substrate, and Mg 2؉ is an obligatory cofactor. The intrinsic Mg 2؉ dissociation constant from metalloenzyme (K M ‫؍‬ 3.5 ؎ 0.3 mM) was experimentally indistinguishable from the sodium pump value. However, the half-maximal concentration for P i binding to metalloenzyme (K P ‫؍‬ 6.3 ؎ 0.6 mM) was significantly higher (ϳ6-fold), and the probability of calcium pump forming phosphoenzyme from bound P i (P c ‫؍‬ 0.04 ؎ 0.03) was significantly lower (ϳ6-fold) than for the sodium pump. From estimates of the rate constants for phosphorylation and dephosphorylation, the calcium pump appears to catalyze phosphoryl group transfer less efficiently than the sodium pump. Ordered binding of Mg 2؉ before P i implies that both calcium pump and sodium pump form a ternary enzyme⅐metal⅐phos-phate complex, consistent with molecular structures of other haloacid dehalogenase superfamily members that were crystallized with Mg 2؉ and phosphate, or a phosphate analogue, bound.The calcium and sodium pumps are classified as P-type, 1 because the energy required for generating ion gradients across cell membranes is derived from ATP by catalyzing hydrolysis in two, Mg 2ϩ -dependent steps (1). The ␥-phosphoryl group is initially transferred to an aspartyl side chain of the protein in one conformation (E 1 ) forming a covalent phosphoenzyme intermediate. Hydrolysis is completed after a conformational change by transferring the phosphoryl group to water. The second step can be reversed by reacting the second conformation (E 2 ), with P i and can be followed either by radioactive 32 P incorporation into the enzyme, or by stable oxygen isotope ( 18 O) exchange between P i and H 2 O. Phosphorylation by P i is a partial reaction that can be uncoupled from other reactions in the pump cycle experimentally, so that the mechanism is simple enough for derivation of the rate equation. Therefore, measurements combining either 32 P incorporation or 18 O exchange with site-directed mutagenesis are potentially a powerful way of identifying amino acids essential for catalyzing phosphoryl group transfer and of learning their function from estimates of intrinsic constants for interaction of reactants with the enzyme.The problem considered in this report is the mechanism of Mg 2ϩ -dependent phosphorylation by P i . The solution is important because the interpretation of observed differences between mutants and wild-type enzyme depends upon the...

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“…Mg2+ is associated with ATP to form the physiological substrate Mg-ATP. Also, Mg2+ is bound to P, to form the phosphorylated intermediate of the Ca"-ATPase (Guillain et al, 1982;Wakabayashi and Shigekawa, 1984;Champeil et al, 1985). In addition to this, the existence of another site for Mg2+ binding has been suggested.…”

Section: Discussionmentioning

confidence: 99%

Localization of a putative magnesium‐binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca²⁺‐ATPase

Girardet

Bally

Arlaud

et al. 1993

European Journal of Biochemistry

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The amino acid sequences of several P-type ATPases share regions of high similarity. The functions of some of these regions, although several proposals have been made, have not yet been absolutely identified. In particular, one of these domains, located within the cytoplasmic loop in the area known as the 'hinge' domain, exhibits the highest degree of conservation. In the sarcoplasmic reticulum Ca2+-ATPase (SERCA-l), this region is located at residues 700-71 2.Comparison of the sequence in this domain with calcium-binding proteins reveals similarities with the center of the helix-loop-helix EF-hand structure that is known to form divalent-cationbinding sites. A 38-residue polypeptide, corresponding to the domain 682-719 of the Ca'+-ATPase was synthesized and tested for its ability to bind divalent cations.Circular-dichroism, intrinsic-fluorescence and tluorescence-energy-transfer studies performed on this polypeptide in solution support the hypothesis that this domain has, in the protein, the ability to bind a divalent cation, presumably Mg", with an affinity of 10-15 mM. This property is observed for the isolated polypeptide in aqueous solvent and in the presence of low concentrations of the a-helix promoter 2,2,2-trifluoroethanol.Substitution of either one or two critical amino acids in the sequence induces a significant reduction of the binding properties.It is proposed that this sequence is involved in the co-ordination of a Mgz+ in the nucleotidebinding site and/or in the phosphorylation site of P-type ATPases.The sarcoplasmic reticulum Ca2+-ATPase is a member of the P-type ATPases family. It is proposed that the Ca2'-ATPase is composed of 10 transmembrane a-helices and a cytoplasmic globular fraction that is divided into two main domains, a small and a large cytoplasmic loop. The large cytoplasmic loop (amino acid residues 330-740) contains the phosphorylation site (Asp351), and most probably the ATPbinding and phosphate-binding sites (McLennan et al., 1985).Several regions of the large loop show a high degree of similarity within P-type ATPases. One of these regions, located around residues 696-707 in the CaZ+-ATPase, the socalled 'hinge' region, presents the highest degree of conservation among the sequences of P-type ATPases (Serrano, 1988) It has been shown that mutations of Thr701, Gly702, Asp703 or Asp707 strongly reduce or totally inhibit the calcium-transport activity of the Ca'+-ATPase (Clarke et al., 1990). The corresponding sequence in NdK-ATPase can be labeled by 5'-(p-fluorosulfonyl)benzoyladenosine, an ATP analogue that inhibits the ATPase activity of this enzyme (Ohta et al., 1986).These observations indicate that the integrity of this part of the protein is essential for the activity of the P-type AT- Pases, and suggest that it may be involved in the binding of ATP to the active site.It has been proposed that the area surrounding the Asp638 of a yeast proton-pumping ATPase (Serrano, 1988;Portillo and Serrano, 1988) could be involved in the binding of the y phosphate of ATP through a chelate...

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Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosine triphosphatase as reflected by organic solvent induced perturbation

Cited by 137 publications

References 45 publications

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Evidence Calcium Pump Binds Magnesium before Inorganic Phosphate

Localization of a putative magnesium‐binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca²⁺‐ATPase

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Interaction of magnesium and inorganic phosphate with calcium-deprived sarcoplasmic reticulum adenosine triphosphatase as reflected by organic solvent induced perturbation

Cited by 137 publications

References 45 publications

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Evidence Calcium Pump Binds Magnesium before Inorganic Phosphate

Localization of a putative magnesium‐binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+‐ATPase

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Product

Resources

About

Localization of a putative magnesium‐binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca²⁺‐ATPase