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The Ca&sup2+;­ATPase from sarcoplasmic reticulum can be inhibited by adenosine 5′-[γ-imidazolidate]triphosphate through the formation of an intramolecular cross­link at the active site which is dependent on the presence of Ca&sup2+; [Bill, E., Gutowski, Z. Bäumert. H. G. (1988). Calcium­dependent inactivation of the Ca&sup2+;­ATPase from sarcoplasmic reticulum by chemically reactive adenosine triphosphate, Eur, J. Biochem, 176, 119–124]. In the present study we show that adenosine 5′-[β­imidazolidate]diphosphate is likewise an inhibitor of the Ca&sup2+;­ATPase effecting a similar inhibition pattern on phosphate release and Ca&sup2+; transport. The overall reaction is Ca&sup2+; dependent and produces a protein band that in SDS/PAGE is indistinguishable from that seen with ATP[imidazolidate]. This shows that the side chain of Asp351 which is claimed to be involved in the cross­linking reaction must be in reach of both the β and theγ phosphate moiety of the respective nucleotides. The cross­linked product is formed by a two-step reaction. The first step is the fast reaction of nucleotide imidazolidate presumably at the phosphorylation site (Asp351) underformation of a mixed anhyride that covalently links nucleotide and protein. Subsequently, the nucleotide is released by a substitution reaction with a second amino acid side chain. This cross­linking reaction is strictly Ca&sup2+; dependent and, remarkably, requires Ca&sup2+; to be added before addition of the inhibitor. It proceeds at two rates and suggests that there are two state of the nucleotide­binding site. This is also supported by the fact that in the absence of Ca&sup2+;, ATP[imidazolidate] reacts only in approximately 50% of the calculated ATP­binding sites (based on 80–90% ATPase of total sarcoplasmic reticulum protein) with no subsequent cross­linking reaction. [ABSTRACT FROM AUTHOR]

1. ATP γP-imidazolidate, synthesized from ATP and carbonyldiimidazole, inhibits the Ca2+-ATPase of sarcoplasmic reticulum in a biphasic manner. A fast first phase is concentration-dependent while a slower second phase is independent of the inhibitor concentration. 2. The inhibition is calcium- and magnesium-dependent. No inhibition occurs in the absence of either cation. 3. Inhibition of the Ca2+-ATPase can be prevented by the protection with ATP. 4. The loss of ATPase activity is pH-dependent. Maximal inhibition coincides with maximal ATPase activity. It indicates a participation of the reacting amino acid side chain in the catalytic cycle. 5. The incorporation of radioactive inhibitor is reversed in a time-dependent fashion while the Ca2+-ATPase remains inhibited. 6. We conclude that ATP γP-imidazolidate initially reacts covalently with an amino acid side chain, probably Asp-351, but is subsequently expelled by a reaction with a second amino acid. 7. This two-step reaction induces an intramolecular cross-link which can be shown by the creation of a new protein band on SDS-PAGE which originates in the Ca2+-ATPase. [ABSTRACT FROM AUTHOR]