Direct photoaffinity labeling of cysteine-295 of alpha-tubulin by guanosine 5'-triphosphate bound in the nonexchangeable site.
The alphabeta-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 beta-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 alpha-tubulin was the major reactive amino acid cross-linked to guanosine by ultraviolet irradiation.
Bai
R
R
Science Applications International Corporation-Frederick, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Choe
K
K
Ewell
J B
JB
Nguyen
N Y
NY
Hamel
E
E
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D003488:Cyanogen Bromide; D003545:Cysteine; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
D000345:Affinity Labels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014404:Tubulin; D006153:Guanosine Diphosphate; C065276:2'-deoxyguanosine 3',5'-diphosphate; D006160:Guanosine Triphosphate; D003545:Cysteine; D003488:Cyanogen Bromide