Benzoylphenylalanine (Bpa), a photoactivatable amino acid was incorporated into the 25 position of the insulin B-chain by a combination of chemical synthesis and enzymatic semisynthesis. Bpa(B25) insulin binds specifically to the insulin receptor with an affinity ≃40% that of native insulin. Addition of biotin or ε-amino-hexanoic acid-iminobiotin at the ε-Lys(B29) position to form Bpa(B25),B29(ε)-biotinyl (BBpa) or Bpa(B25),B29(ε)-(Aha- iminobiotin) analogues had little adverse effect on receptor binding affinity and provided a convenient handle for affinity purification, gel shift assays, and blotting of cross-linked complexes with avidin. The analogues were readily 125I-iodinated with the majority of 125I being incorporated at the Tyr(A14) position; the monoiodo-A14 derivative was easily separated from other forms by high performance liquid chromatography. Photolysis of a complex of the insulin receptor and either Bpa(B25) insulin or [125I]iodo- Tyr(A14),Bpa(B25) insulin yields a covalent insulin-receptor complex. The efficiency of cross-linking with these reagents was unusually high, ranging from 60 to 100%. Furthermore, cross-linking to the insulin receptor results in kinase activation in vitro, and in intact cells insulin receptor phosphorylation and internalization were both activated. Notably, even at saturating concentrations one molecule of Bpa(B25) insulin covalently cross- linked each α2β2 receptor, demonstrating that holoreceptor activation occurs with one high affinity insulin binding site occupied and, if a second insulin binds with lower affinity it must be in a different orientation. Bpa insulin analogues form a new class of photoaffinity reagents which facilitate studies relating insulin-insulin receptor structure and function.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Jan 1 1993|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology