Enzyme phosphorylation with inorganic phosphate causes calcium dissociation from sarcoplasmic reticulum adenosine triphosphatase

Meis, L de; Inesi, Giuseppe

doi:10.1021/bi00325a017

Cited by 21 publications

(6 citation statements)

References 26 publications

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“…*E (which is formed in back-inhibited vesicles) is able to bind neither Pi nor ATP. Thus the enzyme is no longer phosphorylated by Pi [37,38], and the secondary activation of ATP hydrolysis arising from the binding of ATP to the regulatory site is abolished [34]. It may be that during steady-state, triphenylphosphine and 3-nitrophenol bind to 2Ca .…”

Section: Discussionmentioning

confidence: 99%

Modification of ATP regulatory function in sacroplasmic reticulum Ca²⁺‐ATPase by hydrophobic molecules

Wolosker

Petretski

Meis

1990

European Journal of Biochemistry

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The effects of the three hydrophobic molecules triphenylphosphine, trifluoperazine and 3-nitrophenol on Ca2+ uptake and ATPase activity in sarcoplasmic reticulum vesicles was investigated. When ATP was the substrate, triphenylphosphine (3 pM) increased the amount of Ca2+ accumulated by the vesicles. At high concentrations triphenylphosphine inhibited Ca2 + uptake. This effect varied depending on the ATP concentration and the type of nucleotide used. With ITP there was only inhibition and no activation of Ca2+ uptake by triphenylphosphine. On the other hand, trifluoperazine inhibited Ca2+ accumulation regardless of whether ATP or ITP was used as substrate. When 5 mM oxalate was included in the medium in order to avoid binding of Ca2+ to the low-affinity &'+-binding sites of the enzyme, both stimulation by triphenylphosphine and inhibition by trifluoperazine were reduced. In leaky vesicles at low Ca2+ concentrations, triphenylphosphine and 3-nitrophenol were competitive inhibitors of ATPase activity at the regulatory site of the enzyme (0.1 -1 mM ATP). A striking difference was observed when both the high-and low-affinity Ca'+-binding sites were saturated. In this condition, triphenylphosphine and 3-nitrophenol promoted a 3 -4-fold increase in the apparent affinity for ATP at its regulatory site.The Ca2+-ATPase from sarcoplasmic reticulum is a membrane-bound protein responsible for the active Ca2 + accumulation that occurs during relaxation of skeletal muscle [l -31. It has been suggested that during the catalytic cycle the enzyme would change its conformation [4 -61, undergoing a hydrophilic -hydrophobic transition [7 -91. In the E form (Fig. l), the enzyme is phosphorylated by ATP and the catalytic site wold have a hydrophilic character. In the *E form, the enzyme is phosphorylated by PI and the catalytic site would have a hydrophobic character [9].In previous papers, we have shown that a variety of hydrophobic molecules competitively inhibit the phosphorylation of the enzyme by PI, and also decreased the net synthesis of ATP during reversal of the Ca2+ pump [lo-121. The effects observed on Ca2+-ATPase were correlated with hydrophobicity of the drugs, indicating that hydrophobic molecules interact with the catalytic site in the *E enzyme form, impairing the entry of PI [l 11. This interaction might be favoured by the hydrophobic environment of the active site in the *E form.Recently, it has been shown that other hydrophobic molecules such as cyclopiazonic acid [13] and nonylphenol [14] strongly decrease the rate of *E -E transition. The authors attributed this effect either by modifications on ATP induced conformational changes [13] or by stabilization of the *E enzyme form [14].In this report, we compare the effects of three hydrophobic compounds, triphenylphosphine, trifluoperazine and 3-nitrophenol on the catalytic cycle of the Ca'+-ATPase. Although all the drugs tested were shown to compete with P, [ l l -121,

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Section: Discussionmentioning

confidence: 99%

Modification of ATP regulatory function in sacroplasmic reticulum Ca²⁺‐ATPase by hydrophobic molecules

Wolosker

Petretski

Meis

1990

European Journal of Biochemistry

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“…1503,1504,1512 Microsomes have been mainly appliedtothestudyofmetabolismofchemicals. 1472,1475,1494,[1516][1517][1518] Although the microsomal data serve as the input for prediction of hepatic metabolism, 1469,1519,1520 the uptake and transport studies in the microsomes focused on physiological molecules, [1521][1522][1523][1524] and the data on chemicals are rather scarce.…”

Section: Endoplasmic Reticulum Vesiclesmentioning

confidence: 99%

Modeling Kinetics of Subcellular Disposition of Chemicals

Baláž

2009

Chem. Rev.

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“…In fact, formation of the phosphorylated intermediate is rapidly followed by (and is required for) internalization and vectorial translocation of 2 mol of bound calcium per mole of enzyme. Furthermore, a reduction of the enzyme affinity for Ca2+ is observed when the catalytic site is occupied by vanadate (Dupont & Bennett, 1982; Medda & Hasselbach, 1983) or P¡ (de Meis & Inesi, 1985), suggesting that the phosphoryl group, and not the adenosine moiety, is involved in the coupling fThis work was supported by grants from the NIH (5-P01 HL27867) and the Muscular Dystrophy Association.…”

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confidence: 99%

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosine triphosphatase

Teruel

Inesi²

1988

Biochemistry

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The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca2+-ATPase of sarcoplasmic reticulum vesicles. We found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in our preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon "back inhibited" by the rise of Ca2+ concentration in the lumen of the vesicles. When the "back inhibition" is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. No secondary activation of acetyl phosphate utilization by the FITC-enzyme was obtained with millimolar nucleotide. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)

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Enzyme phosphorylation with inorganic phosphate causes calcium dissociation from sarcoplasmic reticulum adenosine triphosphatase

Cited by 21 publications

References 26 publications

Modification of ATP regulatory function in sacroplasmic reticulum Ca²⁺‐ATPase by hydrophobic molecules

Modification of ATP regulatory function in sacroplasmic reticulum Ca²⁺‐ATPase by hydrophobic molecules

Modeling Kinetics of Subcellular Disposition of Chemicals

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosine triphosphatase

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Enzyme phosphorylation with inorganic phosphate causes calcium dissociation from sarcoplasmic reticulum adenosine triphosphatase

Cited by 21 publications

References 26 publications

Modification of ATP regulatory function in sacroplasmic reticulum Ca2+‐ATPase by hydrophobic molecules

Modification of ATP regulatory function in sacroplasmic reticulum Ca2+‐ATPase by hydrophobic molecules

Modeling Kinetics of Subcellular Disposition of Chemicals

Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosine triphosphatase

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Modification of ATP regulatory function in sacroplasmic reticulum Ca²⁺‐ATPase by hydrophobic molecules

Modification of ATP regulatory function in sacroplasmic reticulum Ca²⁺‐ATPase by hydrophobic molecules