Grover W. Everett

Evidence that the tightly bound magnesium in tubulin is associated with the N‐site GTP

In an attempt to determine whether the tightly bound Mg2+ found in purified tubulin in associated with the N‐site GTP or the E‐site GDP or GTP, we removed the E‐site nucleotide by several means: (i) alkaline phosphatase treatment; (ii) displacement using excess GMPPCP; and (iii) polymerizing tubulin in the presence of alkaline phosphatase and non‐hydrolyzable analogues. The Mg2+ content remained equal to about 1 tubulin under conditions where denaturation did not occur. Moreover, the Mg/GTP ratio always remained equal to 1. These results indicate that the Mg2+ is associated with the N‐site GTP.

GTP hydrolysis by tubulin occurs with water oxygen incorporation into inorganic phosphate

Tubulin assembly was conducted in [18O]H2O and the resulting mixture of GTP, GDP and Pi was examined by 31P-NMR. Two Pi signals, separated by about 0.02 ppm, were observed. By combining this mixture with a solution of Pi containing all five possible 16O and 18O isotopomers of Pi, it was shown that the two signals were due to [16O4]- and [16O3, 18O]Pi. The amount of 18O incorporated into the Pi was that expected if the hydrolysis of GTP during tubulin assembly occurs with cleavage of the gamma-phosphorus-bridge oxygen bond.

Complexes of amine cations with lasalocid A, a microbial ionophore

Abstract Complexes having the stoichiometry [R-NH3+][lasalocid A-] have been prepared and characterized, where R represents a series of organic substituents with diverse steric and electronic properties. The crystalline complexes dissolve readily in solvents of low polarity such as chloroform and are characterized in this solvent by molecular weight data and 13C nmr spectroscopy. NMR data indicate that the cations bind the lasalocid A anion via hydrogen bonds to O3, O6, and O8. Molecular weight data show that none of these complexes are appreciably dissociated in chloroform solution.

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.