Affinity labeling of the exchangeable nucleotide binding site in platelet tubulin.
The exchangeable nucleotide binding site of platelet tubulin was labeled with [14C]p-fluorosulfonyl benzoylguanosine (FSBG). FSBG promoted polymerization of tubulin but depolymerization did not occur in the presence of this nucleoside analogue. GTP was able to block FSBG binding to tubulin. [14C]Iodoacetamide-treated tubulin which was first reacted with FSBG was digested with trypsin. The resultant peptides were analyzed by reverse phase high pressure liquid chromatography. One FSBG-labeled peptide could be identified both by its radioactivity and the characteristic UV absorbance spectrum associated with it. This may represent the exchangeable nucleotide site. A second peptide with a distinct nucleotide absorbance peak was found both in FSBG-treated and untreated tubulin preparations. This evidence is suggestive of the non-exchangeable nucleotide binding site.
D000345:Affinity Labels / Q000378:metabolism*; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002250:Carbon Radioisotopes; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007460:Iodoacetamide / Q000097:blood; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin; D014404:Tubulin / Q000097:blood*
C017320:5'-(4-fluorosulfonylbenzoyl)guanosine; D000345:Affinity Labels; D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D014404:Tubulin; D006151:Guanosine; D014357:Trypsin; D007460:Iodoacetamide