Correlation between Ca2+ uptake, Ca2+ efflux and phosphoenzyme level in sarcoplasmic-reticulum vesicles.
Previously [Inesi & de Meis (1989) J. Biol. Chem. 264, 5929-5936] it was shown that dimethyl sulphoxide (Me2SO) increases the amount of Ca2+ accumulated by sarcoplasmic-reticulum vesicles. This effect was related to a decrease in the enzyme affinity for ADP from less than 20 microM to 1 mM. In the present work the apparent affinity of the ADP-sensitive phosphoenzyme for ADP was determined by measuring the rate of ATP synthesis in vesicles previously loaded with Ca2+, at different pH values and in the presence and absence of Me2SO (20%, v/v) and KCl. In all conditions tested, addition of Me2SO never promoted an increase of the apparent Km for ADP to a value higher than 25 microM. ADP inhibits the phosphorylation of the enzyme by Pi. Two components, with Ki values of 80 microM and 8 mM, were detected when vesicles previously loaded with Ca2+ were used. The high-affinity component (Ki 80 microM) was abolished after addition of Me2SO to the medium. Empty vesicles, on the other hand, exhibited only the low-affinity component (Ki 8 mM). During ATP synthesis in a totally aqueous medium, there was a decrease in the phosphoenzyme formed by Pi, after addition of 80-100 microM-ADP to the medium. In the presence of Me2SO this decrease was smaller. The sum of the fractions of phosphoenzyme formed by ATP and by Pi during Ca2+ uptake was higher in the presence of Me2SO than in a totally aqueous medium. Me2SO decreased the passive efflux of Ca2+ measured in the presence of 0.1 mM-Pi and 0.1 mM-MgCl2. In a totally aqueous medium the same decrease was observed when Pi and MgCl2 concentrations were raised to 4 mM. These data suggest that ADP binds to two different enzyme conformations. The binding to one of these conformations (*E) inhibits the phosphorylation of the enzyme by Pi, increases the efflux of Ca2+ and decreases the amount of Ca2+ accumulated by the vesicles.
Benech
J C
JC
Departamento de Bioquimica Instituto de Ciencias Biomedicas Universidade Federal do Rio de Janeiro, Cidade Universitaria, Brasil.
Galina
A
A
de Meis
L
L
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D002118:Calcium; D004121:Dimethyl Sulfoxide