Analysis of the binding sites of Gd3+ on Ca(2+)-transporting ATPase of the sarcoplasmic reticulum through its effects on fluorescence of tryptophan residues and a covalently attached fluorescent probe N-(1-anilinonaphthyl-4)maleimide.
Interaction of Ca2+ and Gd3+ ions with Ca(2+)-transporting ATPase of the sarcoplasmic reticulum (SR-ATPase) was analyzed. Binding of Ca2+ to the transport site caused an enhancement of intrinsic fluorescence of SR-ATPase. Gd3+ also induced fluorescence enhancement. However, the effects of Ca2+ and Gd3+ were additive rather than competitive, indicating that the Gd(3+)-binding site responsible for this enhancement is distinct from the Ca(2+)-transport site. Gd3+ ions at concentrations higher than 10 microM caused a marked fluorescence quenching, indicating an additional interaction at low-affinity binding sites. Interaction of Ca2+ with the transport site led to a quenching of fluorescence of N-(1-anilinonaphthyl-4)maleimide (ANM) covalently attached at SHN [as defined in Yasuoka-Yabe, K. & Kawakita, M. (1983) J. Biochem. 94, 665-675]. In this case the effects of Ca2+ and Gd3+ were mutually exclusive, indicating that Ca2+ and Gd3+ were competing for the same binding site (i.e. the transport site) to affect ANM fluorescence. Competition between Ca2+ and Gd3+ for the Ca(2+)-transport site was also demonstrated by direct measurement of Ca(2+)-binding using nitrocellulose membrane filters. Affinity of Gd3+ for the Ca(2+)-transport site was a little lower than that of Ca2+. Based on these results it was concluded that Gd3+ has at least three kinds of binding sites on SR-ATPase, namely the Ca(2+)-transport site, the Gd(3+)-specific high-affinity site, and a number of low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Imamura
Y
Y
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo.
Kawakita
M
M
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D005456:Fluorescent Dyes; D005682:Gadolinium / Q000378:metabolism*; D007202:Indicators and Reagents; D008301:Maleimides / Q000737:chemistry; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008301:Maleimides; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D014364:Tryptophan; D005682:Gadolinium; D000252:Calcium-Transporting ATPases; D002118:Calcium