Kinetic characterization of detergent-solubilized sarcoplasmic reticulum ATPase

Kosk‐Kosicka, Danuta; Kurzmack, M.; Inesi, Giuseppe

doi:10.1021/bi00279a037

Cited by 101 publications

(80 citation statements)

References 47 publications

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“…Since this interconversion is at the heart of the free energy coupling process [15,23], our results suggest that the molecular machinery for coupling is intact in the monomeric state. ATP synthesis from MgE' -P can also be carried out in the monomeric state [ 10,12,20]. The result is important for mechanistic considerations, because it shows that free energy coupling does not require protein-protein interaction, but it does not necessarily imply that the actual functional unit in the native SR membrane is monomeric.…”

Section: Discussionmentioning

confidence: 99%

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Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Martin

Tanford

1984

FEBS Letters

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Phosphorylation (by inorganic phosphate) of sarcoplasmic reticulum Ca pump protein has been studied in a detergent solution in which the protein has been previously shown to exist as a monomer. The course of the reaction is qualitatively similar to that observed for membrane-bound (possibly oligomeric) protein.In particular, the results indicate that alternation between the two principal conformational states of the Ca pump protein persists in the monomeric state, which suggests that the machinery for coupling of ATP hydrolysis to Ca *+ transport is intact. There are quantitative differences between monomeric and membrane-bound protein with respect to phosphorylation, but they are not necessarily related to the state of association.

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Section: Discussionmentioning

confidence: 99%

“…An early attempt to phosphorylate the solubilized protein with Pi proved unsuccessful [19], but authors in [20] were able to form monomeric MgE'-P in CrzEg in the presence of high concentrations of dimethyl sulfoxide. In our hands, dimethyl sulfoxide was not required.…”

Section: Methodsmentioning

confidence: 99%

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Martin

Tanford

1984

FEBS Letters

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“…Octaethylene glycol mono-n-dodecyl ether (C~2E8) was from Nikko, Japan. Coupling of bovine calmodulin to Sepharose was performed in accordance with Pharmacia instructions, as described earlier [16].…”

Section: Methodsmentioning

confidence: 99%

“…The CaZ+-ATPase was isolated from human packed red cells purchased from local Red Cross, Red cell ghost membrane proteins were solubilized in the presence of the nonionic detergent CI2Es, and the Ca2÷-ATPase was purified by calmodulin affinity chromatography as described previously [16]. The enzyme was stored at -80°C in elution buffer containing 10 mM Tris-maleate, pH 7.4, 130 mM KC1, 0.5 mM MgC12, 5 mM EGTA, 20% glycerol, 750/zM Ct2Es, 2 mM dithiothreitol, and 0.02% of sonicated suspension of egg yolk phosphatidylcholine.…”

Section: Purification Of Cae+-atpasementioning

confidence: 99%

Self‐association of plasma membrane Ca²⁺‐ATPase by volume exclusion

et al. 1995

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At enzyme concentrations above 40 nM the configuration of the purified plasma membrane Ca2+-ATPase is that of calmodulin-insensitive dimers. Dilution of the enzyme generates progressively higher proportions of calmodulin-sensitive monomers with lower V~ and Ca z+ sensitivity than the dimeric enzyme. Dimerization from monomeric state had not been documented before. We investigated whether concentration by volume exclusion, obtained by addition of a large molecular weight dextran to a monomeric Ca2+-ATPase would elicit dimer-like behavior. Dextran induced self-association of monomers, as monitored by fluorescence energy transfer, but the Ca 2+ sensitivity of the re-associated monomers was lower than that of the native dimers. These results suggest that the self-association reaction is structurally but not functionally reversible, and also document the existence of a hitherto unknown kinetic state of the oligomerized Ca2+-ATPase, with high Vmax but low Ca2÷-sensitivity.

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“…At substrate concentrations saturating a high affinity K,,, in the micromolar regime, the maximum level of E-P (4-5 nmol . mg-*) is formed [ 1,2]. The further increase in enzyme activity noted at higher ATP concentrations exhibits a dissociation constant, K,, of several hundred micromolar.…”

Section: Introductionmentioning

confidence: 99%

Sarcoplasmic reticulum CaATPase: product inhibition suggests an allosteric site for ATP activation

Coll

Murphy

1985

FEBS Letters

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Sarcoplasmic reticulum CaATPase hydrolysis of high concentrations of ATP was studied in the presence of ADP. The results obtained were best described as noncompetitive inhibition; added product lowered the V,,,,, but did not affect the slopes of Eadie-Hofstee plots. At these concentrations (OS-5 mM), ATP is known to act as both a substrate and as an activator of turnover. The inability of ATP to overcome ADP inhibition suggests that activating ATP binds to an allosteric regulatory site rather than to the phosphorylated active site.

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Kinetic characterization of detergent-solubilized sarcoplasmic reticulum ATPase

Cited by 101 publications

References 47 publications

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Self‐association of plasma membrane Ca²⁺‐ATPase by volume exclusion

Sarcoplasmic reticulum CaATPase: product inhibition suggests an allosteric site for ATP activation

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Kinetic characterization of detergent-solubilized sarcoplasmic reticulum ATPase

Cited by 101 publications

References 47 publications

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Solubilized monomeric sarcoplasmic reticulum Ca pump protein

Self‐association of plasma membrane Ca2+‐ATPase by volume exclusion

Sarcoplasmic reticulum CaATPase: product inhibition suggests an allosteric site for ATP activation

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Self‐association of plasma membrane Ca²⁺‐ATPase by volume exclusion