Bai R, Ewell J B, Nguyen N Y, Hamel E
Science Applications International Corporation-Frederick, NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702, USA.
J Biol Chem. 1999 Apr 30;274(18):12710-4. doi: 10.1074/jbc.274.18.12710.
Tubulin with [8-14C]GDP bound in the exchangeable site was exposed to ultraviolet light, and radiolabel was cross-linked to two peptide regions of the beta-subunit. Following enrichment for peptides cross-linked to guanosine by boronate chromatography, we confirmed that the cysteine 12 residue was the major site of cross-linking. However, significant radiolabel was also incorporated into a peptide containing amino acid residues 206 through 224. Although every amino acid in this peptide except cysteine 211 was identified by sequential Edman degradation, implying that this was the amino acid residue cross-linked to guanosine, radiolabel at C-8 was usually lost during peptide processing (probably during chromatography at pH 10). Consequently, the radiolabeled amino acid could not be unambiguously identified.
将结合在可交换位点的带有[8-¹⁴C]GDP的微管蛋白暴露于紫外线下，放射性标记与β亚基的两个肽区域发生交联。通过硼酸酯色谱法富集与鸟苷交联的肽后，我们证实半胱氨酸12残基是主要的交联位点。然而，大量放射性标记也掺入了一个包含206至224位氨基酸残基的肽中。尽管通过连续的埃德曼降解鉴定了该肽中除半胱氨酸211外的每个氨基酸，这意味着该氨基酸残基与鸟苷交联，但在肽处理过程中（可能是在pH 10的色谱过程中），C-8处的放射性标记通常会丢失。因此，无法明确鉴定出放射性标记的氨基酸。
