The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements.

Andersen, Johannes Lund; Møller, Jesper; Jørgensen, Peter Leth

doi:10.1016/s0021-9258(18)34331-x

Cited by 130 publications

(33 citation statements)

References 49 publications

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“…The intensity of the attached probe is proportional to the inhibition, which is consistent with earlier measurements (Mitchinson et al, 1982;Andersen et al, 1982;Highsmith, 1984). Complete inhibition is obtained with 5.2-5.3 nmol of FITC/mg of SR protein (Highsmith & Murphy, 1984; Highsmith, 1984).…”

Section: Resultssupporting

confidence: 91%

“…Moreover, the linearity of the data in Figure 1 suggests that all the FITC is bound in a homogeneous environment. These results strengthen the conclusion that FITC stoichiometrically modifies the CaATPase (Mitchinson et al, 1982;Andersen et al, 1982). The data in Figure 2 also indicate that the bound FITC is in an environment that is partially protected from the bulk solvent, in agreement with results from recent Stern-Volmer quenching studies (Highsmith, 1986).…”

Section: Resultssupporting

confidence: 90%

“…Furthermore, the dependence of the monomer activity on free calcium (Murphy et al, 1982;Kosk-Kosicka et al, 1983;Scofano et al, 1985) is cooperative, as is Ca2+ binding to vesicular CaATPase (Inesi et al, 1980;Highsmith, 1982;Scofano et al, 1985). The feasibility of the monomer as the pump, based on its various properties, has been studied in detail by Moller and his associates (Moller et al, 1980(Moller et al, ,1982Andersen et al, 1982Andersen et al, ,1985Vilsen & Andersen, 1986), and a strong argument has been made in favor of a monomeric pump. The drawback to conclusions based on results from detergent-solubilized monomers is that these monomers cannot actually pump, since there is no membrane to provide compartments.…”

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confidence: 99%

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Spatial organization of calcium ATPase molecules in sarcoplasmic reticulum vesicles

Highsmith¹,

Cohen²

1987

Biochemistry

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Fluorescence intensity, polarization, and (Ca2+-Mg2+)-ATPase (CaATPase) activity were measured for sarcoplasmic reticulum (SR) CaATPase with varying amounts of fluorescein isothiocyanate (FITC) attached at a specific site at or near the ATP binding site. The stoichiometry of attached FITC was proportional to the inhibition of ATPase activity, consistent with the independent labeling of one FITC site per CaATPase molecule. Polarization measurements on vesicular CaATPase indicated the occurrence of energy-transfer depolarization that increased as the fraction of binding sites labeled by FITC increased. Addition of the nonionic detergent dodecyl nonaoxyethylene alcohol (C12E9) eliminated the energy-transfer depolarization for all degrees of labeling with little direct effect on the attached FITC molecule. Fluorescence polarization measurements on sizing-column-purified FITC-labeled CaATPase in the presence of 30 mM C12E9 indicated that the sample consisted of homogeneous monomeric CaATPase. The attached FITC molecule was not sensitive to the bulk viscosity for either the vesicular or the detergent-solubilized CaATPase. The midpoints of the transition from vesicular to monomeric CaATPase as a function of increasing detergent concentration were determined from fluorescence polarization and light-scattering measurements. The dependence of these midpoints on the CaATPase concentration indicated a stoichiometry of 262 +/- 35 molecules of C12E9 per CaATPase in the detergent-protein complex. Both measurements gave the same result. The decrease of fluorescence polarization with increasing saturation of the FITC binding sites for vesicular and detergent-solubilized CaATPase was analyzed in terms of energy-transfer depolarization to determine the spatial arrangements of CaATPase molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultssupporting

confidence: 91%

Section: Resultssupporting

confidence: 90%

mentioning

confidence: 99%

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Spatial organization of calcium ATPase molecules in sarcoplasmic reticulum vesicles

Highsmith¹,

Cohen²

1987

Biochemistry

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“…site on the Ca2+-ATPase, the response of the two probes to conformational changes in the Ca2+-ATPase is different. The pH and temperature dependence of the effect of Na3VO4 and Ca2" on the fluorescence of FITC covalently bound to Ca-ATPase is consistent with the two major conformations of the Ca-ATPase predicted by kinetic studies (Pick and Karlish, 1982;Andersen et al, 1982). The E2 conformation corresponds to the state of high fluorescence, and it is favored by EGTA + Na3VO4, low SR vesicles were labeled with FITC (7.5 nmol/mg protein) at 2 mg/ml protein concentration, as described in Methods.…”

Section: Effects Of Tryptic and Peptic Proteolysissupporting

confidence: 77%

Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum

Papp¹,

Pikula²,

Martonosi³

1987

Biophysical Journal

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Ca2+-ATPase molecules were labeled in intact sarcoplasmic reticulum (SR) vesicles, sequentially with a donor fluorophore, fluorescein-5'-isothiocyanate (FITC), and with an acceptor fluorophore, eosin-5'-isothiocyanate (EITC), each at a mole ratio of 0.25-0.5 mol/mol of ATPase. The resonance energy transfer was determined from the effect of acceptor on the intensity and lifetime of donor fluorescence. Due to structural similarities, the two dyes compete for the same site(s) on the Ca2+-ATPase, and under optimal conditions each ATPase molecule is labeled either with donor or acceptor fluorophore, but not with both. There is only slight labeling of phospholipids and other proteins in SR, even at concentrations of FITC or EITC higher than those used in the reported experiments. Efficient energy transfer was observed from the covalently bound FITC to EITC that is assumed to reflect interaction between ATPase molecules. Protein denaturing agents (8 M urea and 4 M guanidine) or nonsolubilizing concentrations of detergents (C12E8 or lysolecithin) abolish the energy transfer. These results are consistent with earlier observations that a large portion of the Ca2+-ATPase is present in oligomeric form in the native membrane. The technique is suitable for kinetic analysis of the effect of various treatments on the monomer-oligomer equilibrium of Ca2+-ATPase. A drawback of the method is that the labeled ATPase, although it retains conformational responses, is enzymatically inactive.

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“…FITC Labeling. Incubation of SR ATPase with FITC results in covalent labeling of the Lys-515 residue (Brandi et al, 1986) and loss of the enzyme's ability to bind and utilize ATP as a substrate (Mitchinson et al, 1982;Andersen et al, 1982). In this case, as well as for the DCCD label (see below), it is possible to digest the protein after the labeling reaction.…”

Section: Resultsmentioning

confidence: 99%

Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosine triphosphatase

Ancos¹,

Inesi²

1988

Biochemistry

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Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the 32P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with [14C]DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis (EP + H2O in equilibrium E + Pi) without significant incorporation of the 14C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with [14C]DCCD under more drastic conditions produced inhibition of calcium binding, 14C radioactive labeling of tryptic fragments A1 and A2 (but not of B), and extensive cross-linking. Intermolecular and, to some extent, intramolecular cross-linking were prevented by exogenous nucleophiles. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A2, and internal cross-linking of fragment A1. It is proposed that both A1 and A2 fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A2 are directly involved in calcium complexation.(ABSTRACT TRUNCATED AT 250 WORDS)

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The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements.

Cited by 130 publications

References 49 publications

Spatial organization of calcium ATPase molecules in sarcoplasmic reticulum vesicles

Spatial organization of calcium ATPase molecules in sarcoplasmic reticulum vesicles

Fluorescence energy transfer as an indicator of Ca2+-ATPase interactions in sarcoplasmic reticulum

Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosine triphosphatase

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