Two Simultaneous Binding Sites for Nucleotide Analogs Are Kinetically Distinguishable on the Sarcoplasmic Reticulum Ca<sup>2+</sup>-ATPase

Mignaco, Julio A.; Lupi, Otilia H.; Santos, Fernanda T.; Barrabin, Héctor; Scofano, Helena M.

doi:10.1021/bi9518353

Cited by 14 publications

(15 citation statements)

References 52 publications

(79 reference statements)

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“…DISCUSSION The affinities of the catalytic-and regulatory nucleotidebinding sites on SERCa1a for ATP (2-5 M and 0.3-1 mM, respectively, (38) are very similar to the K m and K H of scallop Ca-ATPase found here. There is much evidence for two separate binding sites for nucleotide on each Ca-ATPase polypeptide chain (8,39,40), with one of the sites functioning as an allosteric regulator site and the other as the active (catalytic) site. Thus, there are two potential nucleotide-binding sites on the scallop Ca-ATPase that have to be taken into consideration with regard to stabilization of the scallop A and B fragments in the E 1 form of the Ca-ATPase by P i , AMP-PNP, AMP-PCP, and ADP.…”

Section: Resultsmentioning

confidence: 99%

“…All three cytoplasmic domains may have to be intimately juxtaposed to form the site in combination with the hinge region. Thus, atomic models suggest that formation of the non-polar cavity in E 2 -P may involve movement of the A domain close to the catalytic site (39). Support for such a structure comes from studies using proteinase K digestion of SERCA1a (56) and chymotryptic digestion of the Na, K-ATPase (57 the E 2 crystal structure of SERCA1a suggests that the N domain may also be involved in the binding of P i , and there is strong evidence that the C-terminal part of the P domain of SERCA1a is needed for P i to bind (60).…”

Section: Resultsmentioning

confidence: 99%

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Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

Ryan

Stokes

Chen

et al. 2004

Journal of Biological Chemistry

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The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E 1 and E 2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E 1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E 2 (Ca 2؉ -free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca 2؉ -dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E 1 form of scallop Ca-ATPase was occupied by P i , AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca 2؉ . These results suggest that occupation of the catalytic site on E 1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of P i on the E 2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E 2 -P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.The sarco(endo)plasmic reticulum ATPases (SERCAs) 1 transport Ca 2ϩ against an electrochemical gradient from the cytoplasm into the intracellular membranous compartment of the sarcoplasmic or endoplasmic reticulum and play a major role in Ca 2ϩ homeostasis in both muscle and non-muscle cells (1, 2). In the course of transporting Ca 2ϩ , these enzymes pass through a number of relatively well defined intermediate biochemical states that can be isolated and studied. These include forms in which the ␤-carboxyl group of a specific aspartyl residue (Asp 351 in the case of rabbit SERCA1a) is present as an acylphosphate mixed anhydride. Much of the experimental data has been interpreted in terms of the enzyme being able to exist in two basic forms: the E 1 state, in which the enzyme possesses high affinity binding sites for Ca 2ϩ and can be phosphorylated by ATP but not by P i , and the E 2 state, which possesses low affinity Ca 2ϩ sites and can be phosphorylated by P i but not by ATP (3).Comparison of the tryptic digestion patterns of rabbit SERCA1a in its Ca 2ϩ -bound and -free forms has provided some of the strongest direct evidence supporting such a model where the enzyme can exist in two fundamentally different conformational states (4 -6). Recent studies have found that the E 1 (Ca 2ϩ ) 2 form of rabbit SERCa1a shows clear structural differences to the enzyme in its vanadate-stabilized Ca 2ϩ -free (E 2 ) state (7-10). In the E 1 form, the Actuator (A) domain is isolated from the nucleotide (N) and phosphorylation (P) domains, whereas in the E 2 form all three domains are clustered together.The Ca-ATPase from the cross-striated part of the adductor muscle of the sea scallop has been the subject of a ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

Ryan

Stokes

Chen

et al. 2004

Journal of Biological Chemistry

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“…On the other hand, in absence of Ca 2+ the enzyme is also able to promote a non-productive, futile hydrolysis of pNPP solely by the E 2 conformer [14]. Chelerythrine clearly inhibited the unproductive pNPP hydrolysis conducted by the E 2 conformer in absence of Ca 2+ (Fig.…”

Section: Resultsmentioning

confidence: 87%

“…It has been demonstrated that protonation of the residues at the empty Ca 2+ binding sites in E 2 -P is mandatory for normal dephosphorylation of E 2 -P, and that mutations at the Ca 2+ binding sites could affect this protonation [7,10]. Besides ATP, SERCA can hydrolyze pseudosubstrates, like p-nitrophenylphosphate (pNPP) [11,12] or acetylphosphate [12,13], and their hydrolysis is also able to sustain Ca 2+ transport [13,14].…”

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confidence: 99%

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

Vieira

Oliveira

Valente

et al. 2015

Archives of Biochemistry and Biophysics

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“…In addition to ATP, the P-type pumps can hydrolyze other phosphate-containing substrates such as para-nitrophenyl phosphate [26,38,39,40], 3-O-methylfluorescein phosphate [41,42], and acetylphosphate [43,44]. We observed that eosin was a potent inhibitor of pNPPase activity (IC 50 = 3.8 ± 0.23 μM; Fig.…”

Section: Eosin Inhibition Of K + Activated Phosphatase Activitymentioning

confidence: 86%

Kinetic characterization of Na,K-ATPase inhibition by Eosin

Ogan

Reifenberger

Milanick

et al. 2007

Blood Cells, Molecules, and Diseases

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Eosin is a probe for the Na pump nucleotide site. In contrast to previous studies examining eosin effects on Na only ATPase, we examined Na,K ATPase and K activated pNPPase activity in red blood cell membranes and purified renal Na,K ATPase. At saturating ATP (3mM) the eosin IC 50 for Na pump inhibition was 19uM. Increasing ATP concentrations (0.2 -2.5 mM) did not overcome eosin-induced inhibition thus eosin is a mixed-type inhibitor of ATPase activity. To test if eosin can bind to the high affinity ATP site, purified Na,K ATPase was labeled with 20 uM FITC. With increasing eosin concentrations (0.1 uM -10 uM) the incorporation of FITC into the ATP site significantly decreases suggesting that eosin prevents FITC reaction at the high affinity ATP site. Eosin was a more potent inhibitor of K activated phosphatase activity than of Na,K ATPase activity. At 5mM pNPP the eosin IC50 for Na pump inhibition was 3.8 ± 0.23 uM. Increasing pNPP concentrations (0.45 -14.5 mM) did not overcome eosin-induced inhibition thus eosin is a mixedtype inhibitor of pNPPase activity. These results can be fit by a model in which eosin and ATP bind only to the nucleotide site; in some pump conformations, this site is rigid and the binding is mutually exclusive and in other conformations, the site is flexible and able to accommodate both eosin and ATP (or pNPP). Interestingly, eosin inhibition of pNPPase became competitive after the addition of C 12 E 8 (0.1%) but the inhibition of ATPase remained mixed.

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Two Simultaneous Binding Sites for Nucleotide Analogs Are Kinetically Distinguishable on the Sarcoplasmic Reticulum Ca²⁺-ATPase

Cited by 14 publications

References 52 publications

Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

Kinetic characterization of Na,K-ATPase inhibition by Eosin

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Two Simultaneous Binding Sites for Nucleotide Analogs Are Kinetically Distinguishable on the Sarcoplasmic Reticulum Ca2+-ATPase

Cited by 14 publications

References 52 publications

Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

Effect of Orthophosphate, Nucleotide Analogues, ADP, and Phosphorylation on the Cytoplasmic Domains of Ca2+-ATPase from Scallop Sarcoplasmic Reticulum

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

Kinetic characterization of Na,K-ATPase inhibition by Eosin

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Two Simultaneous Binding Sites for Nucleotide Analogs Are Kinetically Distinguishable on the Sarcoplasmic Reticulum Ca²⁺-ATPase