Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells

Schatzmann, H. J.

doi:10.1113/jphysiol.1973.sp010403

Cited by 336 publications

(91 citation statements)

References 25 publications

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“…However, according to their results, ϳ1.5 mol of Ca 2ϩ are occluded per mole of enzyme. This value is in contrast with the proposed stoichiometry of 1 Ca 2ϩ transported per ATP hydrolyzed by the PMCA (20,21,22). In our hands 4 (not shown, but see Ref.…”

Section: Ratio Cacontrasting

confidence: 76%

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Ferreira-Gomes

González-Lebrero

Fuente

et al. 2011

Journal of Biological Chemistry

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In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca 2؉ -ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca 2؉ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca 2؉ concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ؎ 2.2 M, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La III (inducing accumulation of phosphoenzyme in the E 1 P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E 1 P-phosphorylated intermediate of the PMCA.The plasma membrane calcium ATPase (PMCA) 3 is a calmodulin-modulated P-type ATPase responsible for the maintenance of low intracellular concentrations of Ca 2ϩ in most eukaryotic cells. It couples the transport of Ca 2ϩ out of cells with the hydrolysis of ATP into ADP and inorganic phosphate. PMCAs consist of a single polypeptide chain of 127,000 to 137,000 Da. Mammalian PMCAs are encoded by four separate genes (PMCA1-4), and additional isoforms are generated via alternative RNA splicing, which augments the number of variants to Ͼ20 (1).The current kinetic model for PMCA function proposes that the enzyme exists in two main conformations, E 1 and E 2 . E 1 has a high affinity for Ca 2ϩ and is readily phosphorylated by ATP, whereas E 2 has a low affinity for Ca 2ϩ and can be phosphorylated by P i . After binding of intracellular Ca 2ϩ to high affinity sites, E 1 can be phosphorylated by ATP with formation of the intermediate E 1 P. After a conformational transition to E 2 P, Ca 2ϩ would be released to the extracellular medium from low affinity sites, followed by the hydrolysis of the phosphoenzyme to E 2 and a new conformational transition to E 1 (Fig. 1) (2). During some stages of the reaction cycle, Ca 2ϩ becomes occluded, i.e. trapped in the enzyme machinery while it is transported from one side to the other side of the membrane. The principal aim of this study is to identify and kinetically characterize the intermediate(s) of the PMCA containing occluded calcium. Evidence for occlusion in Na,K-ATPase and sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA) has been well established (3), and a good deal of information exists about the occlusion and deocclusion steps of the transported cations in these pumps. Na ϩ and K ϩ are occluded in the E ...

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Section: Ratio Cacontrasting

confidence: 76%

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Ferreira-Gomes

González-Lebrero

Fuente

et al. 2011

Journal of Biological Chemistry

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“…Calcium transport was conducted by the membrane filtration technique (2) in a total volume of 0.5 ml containing 50 mM Tris-Mes (pH 7.5), 50 mM KCI, 5 Estimation of Ionized Calcium Concentration. The ionized calcium concentration was calculated using an EGTA buffer and successive approximation analysis as described by Schatzmann (25). The stability constant for the Ca24-EGTA complex used for calculations was 10"°' and the protonization values a, and a2 were 109.38 and 10877, respectively, giving an association constant at pH 7.5 of 107.83 (20).…”

Section: Methodsmentioning

confidence: 99%

Characterization of Ca²⁺ Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

Buckhout

1984

Plant Physiol.

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The characteristics of Ca2" transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca2 and ATP needed for halfmaximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.

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“…For determination of kinetic parameters, Ca-EGTA buffers were used. Concentrations of free CaZf at pH 6.8 were calculated with an apparent stability constant for Ca-EGTA of 105.94 according to Schatzmann [37].…”

Section: Preparation Of Surcoplusmic Reticulum Vesiclesmentioning

confidence: 99%

Molecular Transformations in Sarcolasmic Reticulum of Fast‐Twitch Muscle by Electro‐Stimulation

Heilmann

Pette

1979

European Journal of Biochemistry

147

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Chronic electro-stimulation of fast-twitch rabbit muscle with the frequency pattern received by a slow-twitch muscle induces a progressive transformation of the sarcoplasmic reticulum. After 2 days stimulation activities of Ca2'-dependent ATPase and of Ca2' transport begin to decrease, and are paralleled by a progressive decrease in CaZi-dependent and CaZi, Mg2' -dependent phosphoprotein formation, reduced rate of dephosphorylation and a rearrangement of the electrophoretic polypeptide and phosphoprotein patterns. These findings suggest a transformation of the sarcoplasmic reticulum to resemble that of a slow-twitch muscle. This transformation is paralleled by increase in time-to-peak of twitch contraction and half relaxation time and occurs before conversion of the myosin light chain pattern is observed. The parallel time course of changes in contractile properties of stimulated muscle and the molecular and functional properties of the sarcoplasmic reticulum emphasizes the definitive role of the latter in determining the twitch characteristics of fast and slow twitch muscles.Sarcoplasmic reticulum has been mainly characterized (for recent reviews see [1,2]) in fast-twitch fibres, since they predominate the fibre populations of most vertebrate muscles. A small number of reports deals with properties of sarcoplasmic reticulum isolated from muscles composed mainly of slow-twitch fibres [3-151. According to these studies, marked differences exist between the sarcoplasmic reticulum in the two fibre types. The present study was undertaken in order to examine whether transformation of fast-twitch muscle as induced by chronic electrostimulation affects the sarcoplasmic reticulum and to which extent changes of the latter can be correlated to the time course of transformation of contractile properties. Changes induced by electro-stimulation should also substantiate the differences existing between the sarcoplasmic reticulum in fast-twitch and slow-twitch muscles [I 51. It has been shown that many properties of fast-twitch muscles can be changed towards those of slow-twitch muscles by indirect stimulation at a frequency pattern resembling that of a motoneuron innervating a slow-twitch muscle [16- Enzymes. ATPase (EC 3.6.1.3); lactate dehydrogenase (EC 1.1.1.27); pyruvate kinase (EC 2.7.1.40); glycogen phosphorylase (EC 2.4.1.1).Symbol. d, days.321. Early stimulation induced changes occur in contractile properties whereas alterations in the myosin are detectable only after prolonged stimulation [21,28,331. Early changes of the sarcoplasmic reticulum have, however, been observed in SrCter's [22,26,29] and our laboratory [25,31,34] and it appeared interesting to study these changes more in detail in order to gain information on their role in the transformation of the overall contractile properties. MATERIALS AND METHODS Stimulation and Physiological MeasurementsMale adult rabbits of the strain 'Weisse Wiener' were stimulated unilaterally via externalized electrodes implanted in close vicinity to the left lateral popliteal ne...

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Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells

Cited by 336 publications

References 25 publications

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Characterization of Ca²⁺ Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

Molecular Transformations in Sarcolasmic Reticulum of Fast‐Twitch Muscle by Electro‐Stimulation

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Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells

Cited by 336 publications

References 25 publications

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

Characterization of Ca2+ Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

Molecular Transformations in Sarcolasmic Reticulum of Fast‐Twitch Muscle by Electro‐Stimulation

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Characterization of Ca²⁺ Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots