TY - JOUR
T1 - Examination of tubulin-nucleotide interactions by protein fluorescence quenching measurements
AU - Karr, Timothy L.
AU - Purich, Daniel L.
N1 - Funding Information:
tRecipient of a NIH Research Career Development Award and an Alfred Foundation Fellowship.
Funding Information:
Guanine nucleotides bind at two distinct sites on the bovine neurotubulin dimer (1). One site is exchangeable with added nucleotide, but the second site will not undergo exchange (2). Recent experiments in our laboratory (3) confirm the idea that GTP hydrolysis is an essential feature of the GTP-supported assembly of microtubules (4-6). Tubulin, charged with [Y-=P]-GTP at the exchangeable site, undergoes assembly and concommitant hydrolysis. Removal of the so-called microtubule associated proteins (MAPS) fraction by phosphocellulose chromatography blocks both processes, and readdition of the MAPS restores both in a coordinated fashion. Nonetheless, the nonhydrolyzable analogues, GMPP(NH)Pa nd GMPP(C&)P, will also support assembly (T-9), and the metabolic significance of hydrolysis is obscured by this observation. Tubules formed in the presence of these analogues have altered response to dilution and to calcium ion-induced depolymerization (9), and the nature of these changes in microtubule properties is a matter of considerable interest. *This research was supported in part by the University of California Cancer Research Coordinating Carmnittee and NIH Biomedical Science Support Grant RR-07099.
PY - 1978/10/30
Y1 - 1978/10/30
N2 - The fluorescence emission spectrum of bovine brain tubulin is quenched upon binding of GTP, GMPP(NH)P, or GMPP(CH2)P to the tubulin·GDP complex. At saturating levels of GTP or its nonhydrolyzable β-γ analogues, the partially quenched spectrum is virtually identical, suggesting that a similar conformational state is attained in each case. Titrations with each ligand yielded dissociation constants of 0.8, 3, and 3μM for GTP, GMPP(NH)P, and GMPP(CH2)P; in all cases the stoichiometry is essentially one molecule of nucleotide per dimer. It is concluded that GDP and GTP stabilize different conformations and that the nonhydrolyzable analogues mimic the binding of GTP. This may be related to the ability of GMPP(NH)P and GMPP(CH2)P to maintain a pre-assembly conformation similar to tubulin·GTP.
AB - The fluorescence emission spectrum of bovine brain tubulin is quenched upon binding of GTP, GMPP(NH)P, or GMPP(CH2)P to the tubulin·GDP complex. At saturating levels of GTP or its nonhydrolyzable β-γ analogues, the partially quenched spectrum is virtually identical, suggesting that a similar conformational state is attained in each case. Titrations with each ligand yielded dissociation constants of 0.8, 3, and 3μM for GTP, GMPP(NH)P, and GMPP(CH2)P; in all cases the stoichiometry is essentially one molecule of nucleotide per dimer. It is concluded that GDP and GTP stabilize different conformations and that the nonhydrolyzable analogues mimic the binding of GTP. This may be related to the ability of GMPP(NH)P and GMPP(CH2)P to maintain a pre-assembly conformation similar to tubulin·GTP.
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U2 - 10.1016/0006-291X(78)91676-5
DO - 10.1016/0006-291X(78)91676-5
M3 - Article
C2 - 728162
AN - SCOPUS:0018176262
VL - 84
SP - 957
EP - 961
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -