TY - JOUR
T1 - Direct photoaffinity labeling of cysteine-295 of α-tubulin by guanosine 5'-triphosphate bound in the nonexchangeable site
AU - Bai, Ruoli
AU - Choe, Kevin
AU - Ewell, John B.
AU - Nguyen, Nga Y.
AU - Hamel, Ernest
PY - 1998/4/17
Y1 - 1998/4/17
N2 - The αβ-tubulin heterodimer has two high affinity guanosine 5- triphosphate binding sites, so that purified tubulin usually contains two molecules of bound guanosine nucleotide. Half this nucleotide is freely exchangeable with exogenous guanine nucleotide, and its binding site has been readily localized to the β-subunit. The remaining nonexchangeable guanosine 5'-triphosphate can only be released from tubulin by denaturing the protein. We replaced the exchangeable site nucleotide of tubulin with 2'- deoxyguanosine 5'-diphosphate, exposed the resulting tubulin to ultraviolet light, degraded the protein, and isolated ribose-containing peptide derived from the nonexchangeable site. A large cyanogen bromide peptide was recovered, and its further degradation with endoproteinase Glu-C established that cysteine-295 of α-tubulin was the major reactive amino acid cross- linked to guanosine by ultraviolet irradiation.
AB - The αβ-tubulin heterodimer has two high affinity guanosine 5- triphosphate binding sites, so that purified tubulin usually contains two molecules of bound guanosine nucleotide. Half this nucleotide is freely exchangeable with exogenous guanine nucleotide, and its binding site has been readily localized to the β-subunit. The remaining nonexchangeable guanosine 5'-triphosphate can only be released from tubulin by denaturing the protein. We replaced the exchangeable site nucleotide of tubulin with 2'- deoxyguanosine 5'-diphosphate, exposed the resulting tubulin to ultraviolet light, degraded the protein, and isolated ribose-containing peptide derived from the nonexchangeable site. A large cyanogen bromide peptide was recovered, and its further degradation with endoproteinase Glu-C established that cysteine-295 of α-tubulin was the major reactive amino acid cross- linked to guanosine by ultraviolet irradiation.
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U2 - 10.1074/jbc.273.16.9894
DO - 10.1074/jbc.273.16.9894
M3 - Article
C2 - 9545331
AN - SCOPUS:0032540292
SN - 0021-9258
VL - 273
SP - 9894
EP - 9897
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 16
ER -