Characterization of the sarcoplasmic reticulum Ca-ATPase from rabbit temporalis muscle.

Gabriel Antonio Sánchez, Daniel Eduardo Di Croce, Ana Clara Casadoumecq, Susana Beatriz Richard, Delia Takara

Biophysics Department, School of Dentistry, University of Buenos Aires, M T de Alvear 2142, 17th Floor B, Buenos Aires 1122AAH, Argentina. gabriel@odon.uba.ar

Archives of oral biology 2012 Oct

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The aim of this work was to isolate the sarcoplasmic reticulum (SR) Ca-ATPase from rabbit temporalis muscle and to determine the optimal conditions for calcium transport and enzymatic activity. SR vesicles were isolated from rabbit temporalis muscle by differential centrifugation, the protein composition analyzed by electrophoresis and compared to fast-twitch muscle membrane suspensions. ELISA was used to determine the sarcoendoplasmic reticulum Ca-ATPase (SERCA) isoform. Ca-ATPase activity was determined by a colorimetric method. Calcium-binding to the Ca-ATPase, calcium uptake, calcium efflux and phosphorylation by P(i) were determined with radioisotopic techniques. Sixty five percent of the total protein concentration of SR membranes suspensions from rabbit temporalis corresponded to SERCA. Of the total SERCA protein, 64% was SERCA 2, 35% was SERCA 1 and less than 1% was SERCA 3. The optimal conditions of the SERCA isolated from rabbit temporalis muscle were: pH 7.2, 5 μM Ca(2+), 100 μM EGTA, 90 μM Mg(2+), 3mM ATP and 100mM KCl and did not differ from fast-twitch skeletal muscle. The temporalis maximal calcium uptake and Ca-ATPase activity were lower but the sensitivity to the specific Ca-ATPase inhibitor thapsigargin was higher. Calcium-binding to the enzyme and calcium efflux were similar while the phosphorylation of the enzyme by P(i) was lower. The lower enzymatic activity and calcium transport capability of the Ca-ATPase isolated from rabbit temporalis, and the higher sensitivity to inhibitory drugs are consistent with the presence of a substantial proportion of SERCA 2, which can be expected in other rabbit masticatory muscles. Copyright © 2012 Elsevier Ltd. All rights reserved.

Citation

Gabriel Antonio Sánchez, Daniel Eduardo Di Croce, Ana Clara Casadoumecq, Susana Beatriz Richard, Delia Takara. Characterization of the sarcoplasmic reticulum Ca-ATPase from rabbit temporalis muscle. Archives of oral biology. 2012 Oct;57(10):1429-37