Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate.
Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.
Nath
J P
JP
Eagle
G R
GR
Himes
R H
RH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006153:Guanosine Diphosphate / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D010777:Photochemistry; D013438:Sulfhydryl Compounds / Q000032:analysis; D014404:Tubulin / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000345:Affinity Labels; D013438:Sulfhydryl Compounds; D014404:Tubulin; D006153:Guanosine Diphosphate; D006157:Guanosine Monophosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004229:Dithiothreitol