Conformational changes of the nucleotide site of the plasma membrane Ca2+-ATPase probed by fluorescence quenching.
Fluorescence quenching by the water-soluble ions I(-) and Cs(+) was used to probe solvent accessibility and polarity of the nucleotide/fluorescein isothiocyanate binding pocket of the purified soluble Ca(2+)-ATPase from plasma membranes. The E(1).Ca.CaM conformer was the least accessible state studied, presenting the lowest suppression constant (K(q)) for both I(-) (K(q) = 6.7 M(-)(1)) and Cs(+) (K(q) = 0.7 M(-)(1)). Accessibility to I(-) was similar for the E(2).VO(4) and E(1).Ca states (K(q) = 7.13 and 7.5 M(-)(1), respectively), whereas E(2) was slightly more accessible (K(q) = 9.1 M(-)(1)). The phosphorylated state E(2)-P presented the highest accessibility, with a K(q) of 16.5 M(-)(1), very near the K(q) of 20.3 M(-)(1) for free FITC. I(-) was unequivocally a better fluorescence quencher, being usually nearly 3-fold as efficient as Cs(+), as indicated by the K(q)(I(-))/K(q)(Cs(+)) ratio (R(q)). The advent of a positive charge cluster on the nucleotide/fluorescein binding pocket in different states was suggested by the increase in R(q), which reached a value as high as 9.5 for the E(1).Ca.CaM conformer. These results indicate (i) a very high water accessibility of the nucleotide/fluorescein pocket for E(2)-P that (ii) is more restricted on the free E(2) state and (iii) becomes rather lower for the E(1).Ca states. Additionally, a positive charge effect of amino acids on the nucleotide site, possibly related to ATP binding and phosphoryl transfer, appears in these E(1).Ca states, being absent in the phosphorylated and nonphosphorylated E(2) states.
Fonseca
Mirian M
MM
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Cidade Universitária, CEP 21941-590, Rio de Janeiro, RJ, Brazil.
Scofano
Helena M
HM
Carvalho-Alves
Paulo C
PC
Barrabin
Hector
H
Mignaco
Julio A
JA
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002586:Cesium / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine
D000814:Aniline Compounds; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007454:Iodides; D008565:Membrane Proteins; D009711:Nucleotides; D009943:Organophosphorus Compounds; D010710:Phosphates; D002586:Cesium; C068798:4-aminophenylphosphate; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate