The interaction of [13C]-enriched colchicine with tubulin as determined by NMR spectroscopy.
[13C]Colchicine, labeled at the tropolone ring methoxy carbon, was used to study interactions with tubulin containing either Mg2+ or Mn2+ at the high affinity divalent cation binding site. Similar experiments were carried out in the presence of excess free divalent cation. The results show that: (1) when Mn2+ occupies the N-site, the 13C signal of the colchicine methoxy carbon of protein-bound colchicine is not broadened, indicating that in protein-bound colchicine the tropolone methoxy group is not close to the N-site cation; (2) when excess Mn2+ is present in solution this 13C signal is severely broadened, indicating that a low affinity divalent cation site or the exchangeable site (E-site) divalent cation is situated near the colchicine binding site; and (3) in the absence of paramagnetic ions a downfield chemical shift is observed for the tropolone methoxy carbon of colchicine upon binding to tubulin, suggesting that colchicine binds near an aromatic group(s) on tubulin.
Osei
A A
AA
Department of Biochemistry, University of Kansas, Lawrence 66045-2106.
Everett
G W
GW
Ringel
I
I
Himes
R H
RH
D000818:Animals; D002247:Carbon Isotopes; D002413:Cations, Divalent; D002417:Cattle; D003078:Colchicine / Q000737:chemistry*; D004347:Drug Interactions; D046911:Macromolecular Substances; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000737:chemistry; D012996:Solutions; D014404:Tubulin / Q000737:chemistry*
D002247:Carbon Isotopes; D002413:Cations, Divalent; D046911:Macromolecular Substances; D012996:Solutions; D014404:Tubulin; D008345:Manganese; D008274:Magnesium; D003078:Colchicine