title: Absorbance and fluorescence properties of 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate bound to coupled and uncoupled Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum.
abstract: Preincubation of skeletal muscle sarcoplasmic reticulum vesicles in the presence of the calcium chelator, [ethylenebis(oxyethylenenitrilo)tetraacetic acid] (EGTA), irreversibly uncouples calcium transport from ATP hydrolysis. Uncoupling cannot be explained by increased membrane permeability, but is associated with decreased capacity of the Ca2+-ATPase to bind noncatalytic, tightly bound ATP and ADP (Berman, M. C. (1982) Biochim. Biophys. Acta 694, 95-121). The effects of EGTA-induced uncoupling on absorbance and fluorescence properties of the bound ATP analog, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), have been studied under static and turnover conditions. Binding of 4.5-4.9 nmol of TNP-ATP/mg, as determined by absorbance difference titration, was relatively unaffected in the uncoupled state. TNP-ATP, bound to coupled vesicles during turnover, showed 6-8-fold enhanced fluorescence and a shift in the difference absorbance maximum from 510 to 493 nm, indicating increased hydrophobicity of the noncatalytic site. Turnover-dependent fluorescence enhancement was diminished by 60-70% in the uncoupled state, while the absorbance maximum wavelength shift was abolished. These data, correlating changes in the environment of the noncatalytic or regulatory nucleotide binding site on the Ca2+-ATPase with coupling activity, indicate that uncoupling is an intramolecular process, involving a ligand binding site on the ATPase, and that exclusion of H2O from the site occupied by noncatalytic nucleotides, during at least part of the catalytic cycle, is an event associated with energy transduction.
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011485:Protein Binding; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium