Affinity labeling of the active site of the Ca2+-ATPase of sarcoplasmic reticulum.
The inactivation of sarcoplasmic reticulum ATPase by fluorescein isothiocyanate (FITC) was shown to have a hyperbolic dependence on the concentration of FITC. The results were quantitatively accounted for by a model in which the reagent first binds reversibly (Kf = 70 microM) to the ATPase and then reacts irreversibly (kmax = 0.8 and 2 min-1 in the absence and presence of 1 mM Mg2+, respectively) to form inactive enzyme. Comparison with the rate constant for the reaction of the model compound alpha-acetyllysine with FITC showed that the FITC-reactive lysyl side-chain of the ATPase is not unusually reactive, indicating that the specificity of the reaction is due to affinity labeling behavior of the reagent. This was supported by protection experiments using ATP, ADP, AdoPP[NH]P, ITP, and TNP-ATP, all of which displayed protection constants similar to their known binding constants to the active site of the ATPase. Both inorganic phosphate and orthovanadate were effective in preventing inactivation by FITC, and calcium only partially reversed the effect of these anions, implying the existence of a ternary complex such as Ca2.E.Pi. Since all ligands (ATP, ADP and Pi) which bind or react at the catalytic site protect it, only the unliganded form appears to bind and react with FITC. Addition of calcium to the MgATP complex of the ATPase caused an increase in the FITC inactivation rate, implying that during turnover there is a larger fraction of unliganded enzyme present, i.e., substrate binding is weaker (Ks is larger). Protection was also observed with fluorescein and two related dyes, eosin and erythrosin. Like FITC, the isothiocyanates of these dyes were effective inactivators. In separate experiments, these two dyes were shown to promote photoinactivation of the ATPase. ATP exerted a protective effect with a concentration dependence consistent with high-affinity active-site binding.
Murphy
A J
AJ
Department of Biochemistry, University of the Pacific, San Francisco, CA 94115.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D008433:Mathematics; D010710:Phosphates / Q000494:pharmacology; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013861:Thiocyanates; D014638:Vanadates / Q000494:pharmacology
D000345:Affinity Labels; D005452:Fluoresceins; D010710:Phosphates; D013861:Thiocyanates; D014638:Vanadates; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate