Interaction of Fluorescein Isothiocyanate with Nucleotide‐Binding Sites of the Ca‐ATPase from Sarcoplasmic Reticulum

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Abstract
We have previously shown that fluorescein 5′ isothiocyanate is a potent inhibitor of the Ca‐ATPase from sarcoplasmic reticulum [Pick, U. and Karlish, S. J. D. (1980) Biochim. Biophys. Acta, 626, 255–261] and completely blocks Ca uptake with ATP but not with acetyl phosphate [Pick, U. and Bassilian, S. (1981) FEBS Lett. 123, 127–130]. Here we show that fluorescein isothiocyanate inhibits in parallel all the ATP‐dependent partial reactions: the Ca‐ATPase, ATP‐dependent Ca uptake and phosphorylation by ATP whereas the phosphorylation reaction by inorganic phosphate is resistant to fluorescein isothiocyanate and Ca uptake with acetyl phosphate is only partly inhibited by the modification.The time course of inactivation by fluorescein isothiocyanate is biphasic and the rate of inactivation is markedly increased at alkaline pH. The observations that a lysine‐specific reagent (2,4,6‐trinitrobenzene sulfonic acid) com‐ petes with binding of fluorescein isothiocyanate and the pH dependence of inactivation strongly suggests binding to a lysine E‐amino group.Mg ions accelerate the rate of inactivation at pH 7.5–8.0 and Ca ions slightly inhibit the inactivation and the binding of fluorescein isothiocyanate.The adenine nucleotides ATP and AdoPP [NH]P protected against inactivation by fluorescein isothiocyanate. A kinetic analysis of the initial rates of inactivation indicates competition between ATP and fluorescein isothiocyanate (the apparent dissociation constant KATP= 38 μM) and suggests a common binding site. Ca ions increase the apparent affinity of the non‐hydrolyzable analog AdoPP [NH]P by an order of magnitude but have no appreciable effect on the apparent affinity of ATP. Mg ions decrease the apparent affinity for adenine nucleotides both in the presence and absence of Ca.It is suggested that (a) fluorescein isothiocyanate binds to and modifies the ATP‐binding site of the enzyme. (b) The enzyme has only one ATP‐binding site but the site exists in two different configurations which sequentially appear at the E2 and the El conformations of the enzymes; and correspond to the ‘regulatory’ ATP‐binding site and to the phosphorylation site and (c) the site is blocked by fluorescein isothiocyanate in both configurations.