Identification of peptides within the base binding domains of the GTP- and ATP-specific binding sites of tubulin.
Using gamma-32P-labeled 8-azidopurine nucleotide photoaffinity probes of GTP and ATP, the respective purine ring binding domain peptides of tubulin have been identified. First, the location of the GTP-specific binding site was shown to be on the beta-subunit, whereas the major ATP-specific binding site was on the alpha-subunit. Using a combination of anion-exclusion and immobilized Al3+ column chromatography, the respective photolabeled tryptic peptides of both nucleotide binding sites were isolated, further purified by reverse phase high performance liquid chromatography (HPLC) and sequenced. Chymotryptic peptides were also generated for the GTP binding site. High retention of the photoinserted radiolabel was observed with many of the peptides on reverse phase HPLC at low flow rates. The stability of the photoinserted radiolabel to HPLC varied with different peptides. However, certain peptides were easily distinguished as being within the base binding domains of the GTP and ATP binding sites of tubulin. Two beta-tubulin peptides containing the majority of photoinserted [gamma-32P]8-azidoguanosine 5'-triphosphate corresponded to N-terminal beta-tubulin amino acid residues 3EIVHIQAGQCGNQIGAK19 and 20FWEVISDEHGIDPTGS35. The peptide containing the majority of photoinserted [gamma-32P]8-azidoadenosine 5'-triphosphate corresponded to the C-terminal alpha-tubulin sequence 431DYEEVGVDSVEGEGEEEGEE450.
Jayaram
B
B
Medicinal Chemistry, College of Pharmacy, Lucille P. Markey Cancer Center, University of Kentucky, Lexington 40536-0093.
Haley
B E
BE
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008868:Microtubule Proteins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010449:Peptide Mapping; D014357:Trypsin; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D008868:Microtubule Proteins; D010446:Peptide Fragments; D014404:Tubulin; C017040:8-azidoadenosine 5'-triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin