Selective inhibition by ionophore A23187 of the enzyme isomerization in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase.

The effect of a lipophilic antibiotic, ionophore A23187, on the purified Ca2+-ATPase from sarcoplasmic reticulum was investigated. When the enzyme was pretreated with A23187 in the presence and absence of Ca2+, the Ca2+-dependent ATPase activity was inhibited almost completely, but the activity of the contaminating Mg2+-ATPase was unaffected. The steady state level of the phosphoenzyme (EP) from ATP or Pi was not substantially altered. When the pretreatment was performed in the presence of Ca2+, EP formation from ATP was only slightly retarded, but EP decomposition was strongly inhibited. Under these conditions, the accumulated EP was ADP-sensitive. EP formation from Pi after chelating of Ca2+ was quite slow, whereas EP once formed was in rapid equilibrium with Pi of the medium. On the other hand, when the pretreatment was performed in the absence of Ca2+, EP formation from ATP was extremely slow, but EP once formed was in rapid dynamic equilibrium with ATP of the medium. EP formation from Pi was very fast, and this EP was in rapid equilibrium with Pi of the medium. These results demonstrate that A23187 selectively inhibits isomerization of the enzyme between the high Ca2+-affinity form and the low Ca2+-affinity form in the catalytic cycle, whether or not the enzyme is phosphorylated. This suggests that interactions between the enzyme protein and the surrounding lipids could play a crucial role in this isomerization.