Spin labelled sarcoplasmic reticulum vesicles: Ca<sup>2+</sup>‐induced spectral changes

Champeil, Philippe; Bastide, Françoise; Taupin, C.; Gary-Bobo, C.M.

doi:10.1016/0014-5793(76)80109-3

Cited by 36 publications

(15 citation statements)

References 17 publications

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“…2 gives prominence to Ca2', through a, because the binding data ( Fig. 1) [9] Thus K' and K' include pair-interaction free energies in addition to the above mentioned free energies. [10] This makes explicit the fact that K' and K' (not K, and K2) are the effective or operational binding constants for a tetramer.…”

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confidence: 98%

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Equilibrium cooperative binding of calcium and protons by sarcoplasmic reticulum ATPase.

Hill¹,

Inesi²

1982

Proc. Natl. Acad. Sci. U.S.A.

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The cooperative equilibrium binding of Ca2+ by sarcoplasmic reticulum ATPase, as modulated by pH, is analyzed by statistical mechanical treatment of a theoretical model. The model consists offour equivalent subunits, in the form ofa square, with nearest-neighbor interactions. Each subunit has one site for binding of one Ca2+ or one proton, but not both. Binding ofeither ligand on a subunit induces a conformational change in the subunit that alters its interaction with its two neighbors. The model gives good agreement with experimental binding data. It should prove useful as a starting point in the analysis of steady-state ATPase activity as a function of Ca2+ and H+ concentrations.Calcium binding is an absolute requirement for activation of sarcoplasmic reticulum (SR) ATPase. Of various classes of binding sites found in SR vesicles (1-4), only a class of high affinity sites is specifically involved in enzyme activation and calcium transport (5-7). Occupation of these sites is accompanied by protein (ATPase) conformational changes, as shown with various spectroscopic methods (8-11).Under equilibrium conditions, the Ca2+ concentration dependence of binding and of Ca2+-induced conformational changes are identical, and the shape ofthe Ca2O titration curves suggests a cooperative binding mechanism (12). In fact, the experimental points obtained at neutral pH were satisfactorily fitted by the use of empirical equations assuming two interacting binding-site domains and were consistent with the notion that two calcium ions are transported per enzyme cycle (13,14). With the more elaborate analysis of the present paper, using a different pCa scale, we find that two interacting domains are not sufficient.Subsequent studies (15) A family of curves displaying calcium binding to SR vesicles as a function of molar Ca2+(c1) and H+(c2) concentrations is shown in Fig. 1. Most of these data were presented previously (12,15). The two different values for maximal binding mentioned in the figure legend reflect the different specific activity of SR preparations over a 3-yr period. The fractional saturation 6 is used as ordinate in the figure.THE MODEL AND ITS PROPERTIES The Ca2+ binding curves in Fig. 1 The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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confidence: 98%

“…Of various classes of binding sites found in SR vesicles (1)(2)(3)(4), only a class of high affinity sites is specifically involved in enzyme activation and calcium transport (5)(6)(7). Occupation of these sites is accompanied by protein (ATPase) conformational changes, as shown with various spectroscopic methods (8)(9)(10)(11).…”

mentioning

confidence: 99%

Equilibrium cooperative binding of calcium and protons by sarcoplasmic reticulum ATPase.

Hill¹,

Inesi²

1982

Proc. Natl. Acad. Sci. U.S.A.

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“…At this point, however, it might be of interest to recall that different lines of evidence have already suggested that binding of nucleotide to Ca 2ϩ -saturated ATPase might result (at least in the case of ATP) in the formation of more than one form of ATPase⅐nucleotide complex (13,18,(37)(38)(39)(40)(41)(42)(43)(44)(45)(46); see further comments in the Supplemental Material).…”

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confidence: 99%

The Average Conformation at Micromolar [Ca2+] of Ca2+-ATPase with Bound Nucleotide Differs from That Adopted with the Transition State Analog ADP·AlFx or with AMPPCP under Crystallization Conditions at Millimolar [Ca2+]

Picard

Toyoshima²,

Champeil³

2005

Journal of Biological Chemistry

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Crystalline forms of detergent-solubilized sarcoplasmic reticulum Ca 2؉ -ATPase, obtained in the presence of either a substrate analog, AMPPCP, or a transition state complex, ADP⅐fluoroaluminate, were recently described to share the same general architecture despite the fact that, when studied in a test tube, these forms show different functional properties. Here, we show that the differences in the properties of the E1⅐AMPPCP and the E1⅐ADP⅐AlFx membraneous (or solubilized) forms are much less pronounced when these properties are examined in the presence of 10 mM Ca 2؉ (the concentration prevailing in the crystallization media) than when they are examined in the presence of the few micromolar of Ca 2؉ known to be sufficient to saturate the transport sites. This concerns various properties, including ATPase susceptibility to proteolytic cleavage by proteinase K, ATPase reactivity toward SH-directed Ellman's reagent, ATPase intrinsic fluorescence properties (here described for the E1⅐ADP⅐AlFx complex for the first time), and also the rates of 45 Ca 2؉ -40 Ca 2؉ exchange at site "II." These results solve the above paradox at least partially and suggest that the presence of a previously unrecognized Ca 2؉ ion in the E1⅐AMPPCP crystals should be re-investigated. A contrario, they emphasize the fact that the average conformation of the E1⅐AMPPCP complex under usual conditions in the test tube differs from that found in the crystalline form. The extended conformation of nucleotide revealed by the E1⅐AMPPCP crystalline form might be only indicative of the requirements for further processing of the complex, toward the transition state leading to phosphorylation and Ca 2؉ occlusion.

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“…Mitochondria1 cytochrome c oxidase catalyses the flow of electrons from cytochrome c to oxygen and H + translocation from the matrix to the cytosolic space [l, 21. Both of these reactions are vectorial in nature as a consequence of the highly asymmetric configuration of the enzyme in the mitochondrial inner membrane.…”

Section: Studies On the Transmembrane Orientation Of Cytochrome C Oximentioning

confidence: 99%

“…These observations and the slow rate of reduction of cytochrome c oxidase by ascorbate alone [20] argue against significant reduction of the oxidase within the time allowed here ( E 5 min). Champeil et al [21] overcame the problem of ascorbate permeability in studies of spin-label sidedness in sarcoplasmic reticulum vesicles by using impermeant NiZ + ions to specifically broaden externally facing spin labels. Further support is given to our measurements of spin-label reduction by the observation that NiS04 causes a very pronounced broadening of the ESR spectrum of the bound 4-maleimido-2,2,6,6-tetramethylpiperidine oxyl.…”

Section: Lipid Head-group Charge On Enzyme Orientation In Reconstitutmentioning

confidence: 99%

Studies on the Transmembrane Orientation of Cytochrome c Oxidase in Phospholipid Vesicles

Casey

Ariano

Azzi

1982

European Journal of Biochemistry

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We report investigations into the direction of orientation of cytochrome c oxidase in reconstituted vesicles and the factors determining this. Measurement of the enzyme orientation employed two independent techniques : monitoring of the level of haem reduction by membrane-permeant and membrane-impermeant reagents and a kinetic analysis of the reduction of a spin label covalently bound to the oxidase surface. The method of preparation of the oxidase vesicles had a pronounced effect on the enzyme orientation and the two measurement techniques agreed in indicating that the proportion of mitochondrially oriented enzyme was approximately 85 and 50 % for vesicles prepared by cholate dialysis and sonication respectively. Our results show that the membrane orientation of the oxidase is determined by interactions between the phospholipid bilayer and the portion of the enzyme embedded therein, as opposed to gross physical constraints. In particular, we demonstrate that the orientation of the oxidase is affected by the fluidity and surface charge of the membrane.Mitochondria1 cytochrome c oxidase catalyses the flow of electrons from cytochrome c to oxygen and H + translocation from the matrix to the cytosolic space [l, 21. Both of these reactions are vectorial in nature as a consequence of the highly asymmetric configuration of the enzyme in the mitochondrial inner membrane. Both at the molecular level using high-resolution electron microscopy [3] and at the subunit level by studies using non-permeant covalent labelling reagents [4,5], it has been shown that the various types of oxidase subunit differ considerably in their exposure at the two sides of the membrane.Following extraction and purification, cytochrome c oxidase can be incorporated into the membrane of phospholipid bilayer vesicles. Such reconstituted cytochrome c oxidase vesicles have been used extensively as a model system for the oxidase in situ in the inner mitochondrial membrane, in particular for studying functions specific to the enzyme; the strength of the reconstituted system in this regard is its freedom from other components of the respiratory chain which may complicate the interpretation of results considerably, particularly when investigating energy-coupling activities. The validity of the reconstituted oxidase vesicles as a model system therefore depends clearly on the oxidase having the same vectorial membrane insertion as in the inner mitochondrial membrane. Surprisingly, however, up to the present time there i\ little reliable evidence that this is the case.Values quoted in the literature for the proportion of reconstituted cytochronie c oxidase having the mitochondrial orientation (i.e. with the cytochrome-c-binding site facing outwards) vary from 50% [6,7] Enzyme. Cytochrome c oxidase (EC 1.9.3.1).of the reconstituted oxidase in the absence and presence of detergents [8 -lo]. It is now well established, however, that the oxidase activity is strongly dependent on the type of detergent employed, which may invalidate this method of measuring oxi...

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Spin labelled sarcoplasmic reticulum vesicles: Ca²⁺‐induced spectral changes

Cited by 36 publications

References 17 publications

Equilibrium cooperative binding of calcium and protons by sarcoplasmic reticulum ATPase.

Equilibrium cooperative binding of calcium and protons by sarcoplasmic reticulum ATPase.

The Average Conformation at Micromolar [Ca2+] of Ca2+-ATPase with Bound Nucleotide Differs from That Adopted with the Transition State Analog ADP·AlFx or with AMPPCP under Crystallization Conditions at Millimolar [Ca2+]

Studies on the Transmembrane Orientation of Cytochrome c Oxidase in Phospholipid Vesicles

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Spin labelled sarcoplasmic reticulum vesicles: Ca2+‐induced spectral changes

Cited by 36 publications

References 17 publications

Equilibrium cooperative binding of calcium and protons by sarcoplasmic reticulum ATPase.

Equilibrium cooperative binding of calcium and protons by sarcoplasmic reticulum ATPase.

The Average Conformation at Micromolar [Ca2+] of Ca2+-ATPase with Bound Nucleotide Differs from That Adopted with the Transition State Analog ADP·AlFx or with AMPPCP under Crystallization Conditions at Millimolar [Ca2+]

Studies on the Transmembrane Orientation of Cytochrome c Oxidase in Phospholipid Vesicles

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Spin labelled sarcoplasmic reticulum vesicles: Ca²⁺‐induced spectral changes