Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

Meissner, Gerhard; Conner, Gregory E.; Fleischer, Sidney

doi:10.1016/0005-2736(73)90355-6

Cited by 471 publications

(193 citation statements)

References 26 publications

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“…Ca2'-ATPase was purified by extraction from SR membranes with a low concentration of deoxycholate according to [13]. SR membranes were treated to remove glycogen phosphorylase as indicated in [7].…”

Section: Methodsmentioning

confidence: 99%

Thermal unfolding of monomeric Ca(II),Mg(II)‐ATPase from sarcoplasmic reticulum of rabbit skeletal muscle

1994

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The thermal unfolding of monomeric and delipidated Ca"-ATPase, solubilized in &Es, can be appropriately described as a non-two-state irreversible denaturation, with only one endothermic peak. In the Ca*+ concentration range (O-O.5 mM) which stimulates the ATPase activity of solubilized monomeric ATPase, Ca2+ shifts the critical temperature midpoint of the denaturation process (r,) from 42 to 50°C without segregation of the endothermic peak into two separate components. Because 20 mM Mg2' only shifts the T, from 42 to 44"C, we conclude that the effect of Car' upon the r,,, is likely to be due to binding to the high affinity Ca2' sites in the ATPase. The effect of Ca2' upon the enthalpy of denaturation is biphasic, suggesting the presence of low affinity Ca 2+ sites (&,, in the millimolar range) in monomeric and solubilized ATPase.

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Section: Methodsmentioning

confidence: 99%

Thermal unfolding of monomeric Ca(II),Mg(II)‐ATPase from sarcoplasmic reticulum of rabbit skeletal muscle

1994

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“…ATPase-enriched vesicles were obtained from the rabbit SR by deoxycholate extraction, using method 2 from [18]. Detergentpurified ATPase [6] was obtained from rabbit SR by dissolving SR vesicles (30 mg/ml) in 3 vol.…”

Section: Methodsmentioning

confidence: 99%

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum

et al. 1982

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Incorporation of 4.5 nmol fluorescein isothiocyanate/mg rabbit sarcoplasmic reticulum, or of 7.4 nmol/ mg purified ATPase, was sufficient to inhibit the activity completely. These results are not consistent with the suggestion (Pick, U. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta 626, 255-261) that 2 mol ATPase were inhibited by each mole of reagent incorporated. A single labelled peptide was purified from the inhibited ATPase and it was shown that Lys 3/190, 10 residues from the N-terminus of tryptic fragment B, was the reactive lysine residue. This site is close to a potential nucleotide-binding fold in the ATPase sequence. A similar peptide showing only 2 conservative replacements was isolated from the sarcoplasmic reticulum of the lobster. (Ca 2 + + Mg 2 + )A TPase inhibition Reactive lysine residue Fluorescein isothiocyanateIntegral membrane protein Nucleotide-binding fold (Sarcoplasmic reticulum)

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“…Caz+-ATPase was purified using deoxycholate by method 2 of Meissner et al (1973), yielding closed vesicles essentially free of extrinsic proteins, in which the Ca2*-ATPase accounts for more than 90% of the total protein. Protein concentration was determined by the method of Lowry et al (1951).…”

Section: Preparation Of Sarcoplasmic Reticulum Vesicles and Purified mentioning

confidence: 99%

Topology of sarcoplasmic reticulum Ca²⁺‐ATPase: An infrared study of thermal denaturation and limited proteolysis

et al. 1998

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Sarcoplasmic reticulum Ca2+-ATF'ase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as a-helix, P-sheet, etc. . . . , two unusual bands, one at 1,645 cm" in H 2 0 buffer and the other at 1,625 cm" in D 2 0 buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm" is tentatively assigned to a-helical segments located in the cytoplasmic domain and coupled to /?-sheet structure, whereas the band at 1,625 cm" arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% a-helical segments in the transmembrane plus stalk domain; 20% a-helix and 22% @sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca*+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm" band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm" band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37"C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.Keywords: Ca2+-ATPase; infrared spectroscopy; protein structure; proteolysis; sarcoplasmic reticulum; thermal analysis Knowledge of the structure-function relationship is essential in understanding the molecular mechanisms underlying the membranecontrolled biological processes. The X-ray three-dimensional structure of membrane proteins is still not well known, except for a few cases. Therefore, other lower resolution spectroscopic methods have to be used to gain insight of the structure and function of membrane proteins. Sarcoplasmic reticulum Ca2+-ATPase is an integral membrane protein that pumps calcium out of the cytoplasm during striated muscle relaxation (Martonosi, 1996). This ATPase is part of a family of P-type ion pumps that includes several cation-activated ATPases having in common ten predicted transmembrane helical segments (Stokes et al., 1994).The structure of sarcoplasmic reticulum Ca2+-ATPase has been predicted from the amino acid sequence (MacLennan et al., 1985) and from electron microscopy observations (Toyoshima et al., 1993). The protein appears to consist of an extensive beak-shaped cytoplasmic domain containing interconnected a-helical and P-strand segments, a stalk connecting the beak with the membrane, and the ten transmembrane segments characteristic of P-type ion pumps (Stokes et al., 1994). The cytoplasmic domain contains the active sites of ATP hydrolysis and phosphorylation while the Ca2+ channel is expected to be associated to the transmembrane domain. Some Ca2+-...

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Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

Cited by 471 publications

References 26 publications

Thermal unfolding of monomeric Ca(II),Mg(II)‐ATPase from sarcoplasmic reticulum of rabbit skeletal muscle

Thermal unfolding of monomeric Ca(II),Mg(II)‐ATPase from sarcoplasmic reticulum of rabbit skeletal muscle

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum

Topology of sarcoplasmic reticulum Ca²⁺‐ATPase: An infrared study of thermal denaturation and limited proteolysis

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Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

Cited by 471 publications

References 26 publications

Thermal unfolding of monomeric Ca(II),Mg(II)‐ATPase from sarcoplasmic reticulum of rabbit skeletal muscle

Thermal unfolding of monomeric Ca(II),Mg(II)‐ATPase from sarcoplasmic reticulum of rabbit skeletal muscle

Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca2+‐dependent adenosine triphosphatase of sarcoplasmic reticulum

Topology of sarcoplasmic reticulum Ca2+‐ATPase: An infrared study of thermal denaturation and limited proteolysis

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Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca²⁺‐dependent adenosine triphosphatase of sarcoplasmic reticulum

Topology of sarcoplasmic reticulum Ca²⁺‐ATPase: An infrared study of thermal denaturation and limited proteolysis