Interaction of spin-labeled nucleotides with sarcoplasmic reticulum adenosine triphosphatase

Abstract

Spin-labeled derivatives of AMP-PCP, ATP, and 2'-deoxy-ATP, with a nitroxide moiety attached to the ribose ring [3'-O-(1-oxy-2,2,5,5-tetramethylpyrroline-3-carbonyl)nucleotide], are used to study the nucleotide binding site stoichiometry of sarcoplasmic reticulum (SR) ATPase. With all derivatives, a maximal binding of 4.5 nmol/mg of SR protein is found, a value close to the number of phosphorylation sites obtained with ATP. The spin-labeled nucleotides cannot be utilized by the enzyme as substrates. Binding of spin-labeled nucleotides is inhibited by labeling the ATPase with fluorescein 5'-isothiocyanate, indicating that all the labeled nucleotide is located at the catalytic site. Additions of spin-labeled ATP to vesicle suspensions during steady turnover demonstrate competitive inhibition of both catalysis and the regulatory effect normally exhibited by ATP. As secondary binding of spin-labeled ATP is not detected at pertinent concentrations, it is suggested that both functions of ATP may be effected through a single site.