Inhibition of rat liver microsomal Ca2+-ATPase by fluorescein-5'-isothiocyanate.

Rat liver microsomal fraction was incubated at pH 8.8 with fluorescein-5'-isothiocyanate in a Tris-buffered sucrose medium. This treatment completely inhibited ATP-dependent Ca2+ transport, Ca2+-ATPase activity, and Ca2+-ATPase phosphoenzyme intermediate formation. Inhibition of Ca2+ transport and phosphoenzyme intermediate formation by fluorescein-5'-isothiocyanate was partially prevented by including ATP in the treatment medium. These data taken together are consistent with the proposal that fluorescein-5'-isothiocyanate binds the Ca2+-ATPase ATP-binding site, suggesting the presence of a lysine residue in this domain. Fluorescein-5'-isothiocyanate labeling of microsomal proteins had no measurable effect on the basal, Mg2+-ATPase activity. Using fluorescein-5'-isothiocyanate-labeled microsomal fraction, we demonstrated that the Mg2+-ATPase activity was inhibited by Ca2+.