Preparation of N epsilon B28-monoazidobenzoyl insulin-like growth factor I and photoaffinity labeling of insulin-like growth factor I receptor.
Recombinant human insulin-like growth factor I (hIGF-I) was reacted with azidobenzoyl hydroxysuccinimide to produce a mixture of photoactive hIGF-I derivatives. The mixture was purified by reversed-phase HPLC to yield three mono-substituted azidobenzoyl hIGF-Is. One of the derivatives was identified by amino acid sequencing as N epsilon B28-monoazidobenzoyl hIGF-I. This derivative was indistinguishable from native hIGF-I when bioassayed in Rat-1 fibroblasts. A 120-kDa band, the alpha subunit of the IGF-I receptor, was specifically labeled in Rat-1 plasma membranes by this photoprobe. The labeling of this band was reduced by hIGF-I at 1 nM and completely abolished by hIGF-I, but not insulin, at 100 nM, indicating the specificity of the photolabeling of the IGF-I receptor by this fully active IGF-I photoprobe.
Yip
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Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Hsu
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Olefsky
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JM
Seely
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D000345:Affinity Labels / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001386:Azides; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D011994:Recombinant Proteins; C029534:hydroxysuccinimidyl-4-azidobenzoate; D007334:Insulin-Like Growth Factor I; D017526:Receptor, IGF Type 1; D013936:Thymidine