A fluorescence investigation of the nucleotide binding sites of the Ca ATPase.
Competitive binding and fluorescence energy transfer experiments were used to examine the binding of 2'[3']-O-(2,4,6-trinitrophenyl) adenosine-5'-diphosphate (TNP-ADP) to the catalytic site of Ca ATPase. Fluorescein isothiocyanate (FITC), which is known to covalently bind near the catalytic site (13), was shown to exclude all TNP-ADP binding. TNP-ADP, in turn, will protect against FITC labeling of the Ca ATPase in its native state and in both phosphoenzyme forms. The competitive nature of these probes indicates that TNP-ADP binds to the catalytic site exclusively. Fluorescence energy transfer studies using TNP-ADP as an energy acceptor and 1,N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) as an energy donor were used to estimate the distance between nucleotide binding sites in the enzyme complex. A lower limit for the distance measured was 44 A. This is interpreted to be the distance between catalytic sites on adjacent monomers of a dimer unit. The results of this work are consistent with a single nucleotide site per ATPase monomer.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism; D008958:Models, Molecular; D009132:Muscles / Q000201:enzymology; D011817:Rabbits; D013861:Thiocyanates / Q000378:metabolism
D005452:Fluoresceins; D013861:Thiocyanates; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate