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Characteristics of the Sarcoplasmic Reticulum Ca²⁺-dependent ATPase from Masticatory Muscles

¹ Biophysics Department, Faculty of Dentistry, University of Buenos Aires, MT de Alvear 2142, 1122 Buenos Aires, Argentina; and
² Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina;

View larger version (15K): [in this window] [in a new window]   Figure 1. ATPase activity of SR membranes from masticatory muscles. SR vesicles (0.1 mg/mL) were incubated at 37°C in media containing 50 mmol/L MOPS-Tris (pH 7.2), 3 mmol/L ATP, 3 mmol/L MgCl2, 100 mmol/L KCl, and 10 µmol/L calcimycin. No Ca2+ was added. The reactions and the ATPase activity determinations were performed as indicated in MATERIALS & METHODS. Error bars indicate SD; n = 4. (A) ATPase activity as a function of EGTA concentration. SR vesicles from masseter (•) and medial pterygoid () muscles were incubated in the above medium with EGTA added as indicated in the abscissa. (B) ATPase activity in the presence of thapsigargin for masticatory muscles. SR vesicles from masseter and medial pterygoid muscles were incubated as described above in the presence of 0.1 mmol/L EGTA and with (hatched bars) or without (blank bars) 0.4 µmol/L thapsigargin.

View larger version (19K): [in this window] [in a new window]   Figure 2. Dependence of Ca-ATPase activity on pH. SR vesicles (0.1 mg/mL) from masseter (•), medial pterygoid (), and fast muscles () were incubated at 37°C in media containing 50 mmol/L MOPS-Tris (pH 6.5–8.5) or MES-Tris (pH 5.0–6.4), 3 mmol/L ATP, 0.1 mmol/L CaCl2, 0.1 mmol/L EGTA, 3 mmol/L MgCl2, 100 mmol/L KCl, and 10 µmol/L calcimycin. The reactions and the Ca-ATPase activity determinations were performed as indicated in MATERIALS & METHODS. Bars, SD; n = 5.

View larger version (28K): [in this window] [in a new window]   Figure 3. Ca-ATPase activity as a function of the substrate and cofactor concentrations. SR vesicles (0.1 mg/mL) from masseter (•), medial pterygoid (), and fast muscles () were incubated at 37°C in a basic medium containing MOPS-Tris (pH 7.2), ATP, MgCl2, KCl, CaCl2, EGTA, and calcimycin modified as follows. (A) Ca-ATPase activity as a function of [Ca2+]. SR vesicles were incubated in the above basic medium except for the addition of various free calcium and EGTA concentrations to render concentrations as indicated in the abscissa. (B) Ca-ATPase activity as a function of [Mg2+]. SR vesicles were incubated in the above basic medium except for the addition of various free magnesium concentrations as indicated in the abscissa. (C) Ca-ATPase activity as a function of [KCl]. SR vesicles were incubated in the above basic medium except for the addition of various KCl concentrations as indicated in the abscissa. (D) Ca-ATPase activity as a function of [ATP]. SR vesicles were incubated in the above basic medium except for the addition of various ATP concentrations as indicated in the abscissa. (A-D) Reactants at constant concentration within each Fig. were as in Fig. 2. The reactions and the Ca-ATPase activity determinations were performed as indicated in MATERIALS & METHODS. Error bars indicate SD; n = 4.

View larger version (15K): [in this window] [in a new window]   Figure 4. Inhibition of Ca-ATPase by thapsigargin. SR vesicles (0.1 mg/mL) from masseter (•), medial pterygoid (), and fast muscles () were incubated at 37°C in media containing 50 mmol/L MOPS-Tris (pH 7.2), 3 mmol/L ATP, 3 mmol/L MgCl2, 100 mmol/L KCl, 0.1 mmol/L CaCl2, 0.1 mmol/L EGTA, and thapsigargin as indicated in the abscissa. (A) Inhibition of Ca-ATPase activity. SR vesicles were incubated in the above medium with 10 µmol/L calcimycin added. The reactions and the Ca-ATPase activity determinations were performed as indicated in MATERIALS & METHODS. Bars, SD; n = 5. (B) Inhibition of ATP-dependent calcium uptake. SR vesicles were incubated in the above medium with the addition of tracer amounts of (45Ca)CaCl2. The reactions were performed as indicated in MATERIALS & METHODS. Bars, SD; n = 4.

Journal of Dental Research, Vol. 83, No. 7, 557-561 (2004)
DOI: 10.1177/154405910408300709


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