Energy-transfer studies of the distance between the high-affinity metal binding site and the colchicine and 8-anilino-1-naphthalenesulfonic acid binding sites on calf brain tubulin.
The high-affinity metal divalent cation Mg2+, associated with the exchangeable guanosine 5'-triphosphate (GTP) binding site (E site) on purified tubulin, has been replaced by the transition metal ion Co2+ on tubulin as well as on the tubulin-colchicine, tubulin-allocolchicine and tubulin-8-anilino-1-naphthalenesulfonic acid (tubulin-ANS) complexes. While pure native tubulin readily incorporated 0.8 atom of Co2+ per tubulin alpha-beta dimer, incorporation was reduced to 0.4 atom of Co2+ per mole of tubulin when it was complexed with colchicine, indicating that the conformational change induced in tubulin by the binding of colchicine leads to a reduced accessibility of the divalent cation binding site linked to the E site without necessarily changing the intrinsic binding constant. The fluorescence emission spectra of tubulin-bound colchicine, allocolchicine, and ANS displayed a strong overlap with the Co2+ absorption spectrum, identifying these as adequate donor-acceptor pairs. Fluorescence energy-transfer measurements were carried out between tubulin-bound colchicine (or allocolchicine) and ANS as donors and tubulin-complexed Co2+ as acceptor. It was found that the distance between the ANS and the high-affinity divalent cation binding sites is greater than 28 A, while that between the colchicine and the divalent cation binding sites is greater than 24 A.(ABSTRACT TRUNCATED AT 250 WORDS)
Ward
L D
LD
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Timasheff
S N
SN
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D003035:Cobalt / Q000378:metabolism*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D014404:Tubulin; D003035:Cobalt; C027132:1-anilino-8-naphthalenesulfonate