Equilibrium and Kinetic Studies of Calcium Transport and ATPase Activity in Sarcoplasmic Reticulum

Inesi, Giuseppe; Kurzmack, M.; Kosk‐Kosicka, Danuta; Lewis, David E.; Scofano, Helena M.; Guimarães-Motta, Horácio

doi:10.1515/znc-1982-7-819

Cited by 38 publications

(19 citation statements)

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“…Deletions of any single residues in the Glu 40 -Ser 48 loop resulted in almost complete loss of the activity, while their nonconservative substitutions caused only partial decrease or rather slight increase in the activity. Single deletions or substitutions of the residues in the adjacent N-terminal (His 32 -Asn 39 ) and C-terminal (Leu 49 -Ile 54 ) regions of the Glu 40 -Ser 48 loop had only slight or moderate effect on the activity, except that the specific substitutions of Asn 39 (N39D and N39T, but not N39A) had a significantly reduced activity. Quadruple alanine substitutions of Asn 39 , Glu 40 , Glu 44 , and Glu 45 were previously shown to cause no loss of function (22), and the present results are in essential agreement.…”

Section: Effects Of Deletions and Substitutions On Atp Hydrolysis-mentioning

confidence: 97%

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

Daiho

Yamasaki

Wang

et al. 2003

Journal of Biological Chemistry

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E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from P i was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca 2؉ -induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu 40 -Ser 48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.Sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA1a) 1 is a representative member of P-type ion transporting ATPases and catalyzes Ca 2ϩ transport coupled with ATP hydrolysis (Fig. 1) (Refs. 1 and 2, and for recent reviews, see Refs. 3 and 4). In the catalytic cycle, the enzyme is activated by binding of two Ca 2ϩ ions (E2 to E1Ca 2 , steps 1 and 2) and then autophosphorylated by MgATP to form ADP-sensitive phosphoenzyme (E1P, step 3). Upon formation of this EP, the bound Ca 2ϩ ions are occluded in the transport sites. The subsequent isomeric transition to ADP-insensitive form (E2P, step 4) will result in a reduction in affinity and a change in orientation of the Ca 2ϩ binding sites, and thus a Ca 2ϩ release into lumen (step 5). Finally, hydrolysis takes place and returns the enzyme into an unphosphorylated and Ca 2ϩ -unbound form (E2, step 6). E2P can also be formed from P i in the presence of Mg 2ϩ and absence of Ca 2ϩ by reversal of its hydrolysis.The enzyme has three cytoplasmic domains (N, P, and A), which are widely separated in the Ca 2ϩ bound form (E1Ca 2 ) and associated in the Ca 2ϩ -unbound and thapsigargin-bound form (E2(TG)) (5, 6) ( Fig. 2). The modeling of tubular crystals formed with decavanadate (E2V) revealed (5) that three cytoplasmic domains gather to form a most compactly organized single headpiece in E2V. With the limited proteolysis experiments, we previously showed (7, 8) that E2V is very similar to E2P in domain organization and that E2P is the intermediate having the most compactly organized headpiece in the catalytic cycle. The results further indicated that a large rotation of A domain (by ϳ90° (5)) and its strong association with P and N domains most likely occur during the E1P to E2P transition and suggested that stabilization energy provided by intimate contacts between all three cytoplasmic domains in E2P will provide energy for moving transmembrane helices and release the bound Ca 2ϩ ions.It is thus crucial to find out structural elements essential for the A domain movement and resulting domain organization and for transmitting these changes to transmembrane helices. We have recently identified the Lys 189 -Lys 205 outermost loop of A domain as...

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Section: Effects Of Deletions and Substitutions On Atp Hydrolysis-mentioning

confidence: 97%

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

Daiho

Yamasaki

Wang

et al. 2003

Journal of Biological Chemistry

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“…There are a number of postulated mechanisms for the Ca-ATPase of skeletal (Inesi et al, 1982;Guimaraes-Motta and DeMeis, 1980;Takakuwa and Kanazawa, 1982) and cardiac (Tada et al, 1980) SR. Each of these mechanisms invokes transitions between intermediates of the pump and many of these transitions involve binding or desorption of ligands. The mechanisms mentioned share the feature that ATP binds after outside calcium, while ADP desorbs before calcium desorbs to the internal com-partment.…”

Section: Introductionmentioning

confidence: 99%

Undirectional calcium and nucleotide fluxes in cardiac sarcoplasmic reticulum. II. Experimental results

Feher¹,

Briggs²

1984

Biophysical Journal

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Unidirectional calcium influx and efflux were evaluated in cardiac sarcoplasmic reticulum (SR) by 45Ca-40Ca exchange at steady state calcium uptake in the absence of calcium precipitating anions. Calcium efflux was partitioned into a pump-mediated efflux and a parallel passive efflux by separately measuring passive efflux referable to the steady state. Unidirectional and net ATP-ADP fluxes were measured using [3H]-ATP----ADP and [3H]-ADP----ATP exchanges. Methods are presented that take into account changing specific activities and sizes of the nucleotide pools during the measurement of nucleotide fluxes. The contribution of competent and incompetent vesicles to the unidirectional and net nucleotide fluxes was evaluated from the specific activity of these fluxes in incompetent vesicles and from the fraction of vesicles that were incompetent. The results indicate that, in cardiac SR, unidirectional calcium fluxes are larger than the unidirectional nucleotide fluxes contributed by competent vesicles. Because the net ATPase rate of competent vesicles is similar to the parallel passive efflux, it appears that cardiac SR Ca-ATPase tightly couples ATP hydrolysis to calcium transport even at static head, with a coupling ratio near 1.0.

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“…The change in the structure of water would increase the activity of the calcium ion such that it could diffuse downhill towards a region of high calcium activity upon the opening of a selective channel. In an alternate view, active transport involves a pump having a different affinity for its transported ion when the binding site is accessible to different sides of the membrane (Inesi et al, 1982;Giumaraes-Motta and DeMeis, 1980;Inesi et al, 1980;Takakuwa and Kanazawa, 1982). The essential difference between these views is that selectivity is provided by a gated channel in the first view and by a binding site in the second, and that free-energy transduction is provided through water in the first and through protein conformation in the second.…”

Section: Introductionmentioning

confidence: 99%

Unidirectional calcium and nucleotide fluxes in sarcoplasmic reticulum. I. Interpretation of flux ratios for different reaction schemes

Feher¹

1984

Biophysical Journal

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The relation between unidirectional calcium and nucleotide fluxes was examined for different ATPase reaction schemes of the sarcoplasmic reticulum. The schemes considered differed in the order of sorption and desorption of calcium, ATP, and ADP. The results suggest that the theoretical relation between calcium and nucleotide fluxes depends on the reaction scheme and that experimental measurements can distinguish among them. The results obtained are generally valid and do not depend on assumptions of equilibrium or pseudoequilibrium between intermediate states of the pump.

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Equilibrium and Kinetic Studies of Calcium Transport and ATPase Activity in Sarcoplasmic Reticulum

Cited by 38 publications

References 0 publications

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

Deletions of Any Single Residues in Glu40-Ser48 Loop Connecting A Domain and the First Transmembrane Helix of Sarcoplasmic Reticulum Ca2+-ATPase Result in Almost Complete Inhibition of Conformational Transition and Hydrolysis of Phosphoenzyme Intermediate

Undirectional calcium and nucleotide fluxes in cardiac sarcoplasmic reticulum. II. Experimental results

Unidirectional calcium and nucleotide fluxes in sarcoplasmic reticulum. I. Interpretation of flux ratios for different reaction schemes

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