Abstract
Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).
Highlights
From the Departmentof Physiology, Uniuersityof Pittsburgh Schoolof Medicine, Pittsburgh, Pennsylvania15261 and the Department of Biochemistry and Biophysics, Universityof Pennsylvania School of Medicine, Philadelphia, Pennsyluania19104
Magnesium transport across sarcoplasmic reticulum required for the hydrolysis of phosphoenzyme formed from (SR) vesicles was investigated in reaction mixtures of ATP and (Ca2+)-ATPawsehich is a necessarystep for enzyme various compositionusing antipyrylazo I11 or arsenazo turnover and further translocationof Ca2+(2, 3)
The concentration and time course of Mg2+released into the medium was measured during ATP-dependent Ca2+uptake by SR.In reaction mixtures containing up to 3 mM Mg2+,small changes in free magnesium of 10 PM or less were accurately detected withoutinterferencefromchanges in free Ca2+of up to 100 MM
Summary
Changes in extravesicular free M e were measured during the Ca2+ transport process, and a second addition of M e was used to recalibrate and verify the linearity of the dye's response to changes in free M%+.Active CaZ+uptake and Mg2+fluxes across the SR membrane were measured in reaction mixtures oflow ionic strength which consisted of 200 mM sucrose, 1mM Mg gluconate, 50 p M Ca gluconate, 50 p~ EGTA, 30 mM imidazole or HEPES, pH 6.87.0, Mg-ATP, or an ATP-regenerating system, at 23 "C.
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