High-yied covalent attachment of epidermal growth factor to its receptor by kinetically controlled, stepwise affinity cross-linking

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Abstract

We report here the use of a stepwise affinity cross-linking technique in the specific covalent attachment of epidermal growth factor (EGF) to its receptor. A heterobifunctional cross-linking reagent, sulfo-TV-succinimidyl 4-(fluorosulfonyl)benzoate (SSFSB), which contains a rapidly reacting sulfo-N-succinimidyl active ester and a much more slowly reacting aromatic fluorosulfonyl moiety, was synthesized and characterized. Murine EGF (mEGF) was modified by the cross-linker to yield as the major product a derivative of mEGF having the (fluorosulfonyl)benzoyl moiety attached covalently at the amino terminus. SSFSB-modified, 125I-labeled mEGF was separated from unreacted SSFSB by size-exclusion chromatography and applied to shed membrane vesicles from A431 human carcinoma cells. Binding of derivatized 125I-mEGF to vesicles led to high yields (>60%) of covalent linkage of 125I-mEGF to the EGF receptor, as determined by measurement of the fraction of specifically bound radiolabel which comigrated with the EGF receptor in NaDodSO4-polyacrylamide gels. The specificity of affinity cross-linking was evident in the negligible degree of labeling of species other than the EGF receptor and in the retention of EGF-stimulated receptor kinase activity after cross-linking.

Original languageEnglish (US)
Pages (from-to)7341-7346
Number of pages6
JournalBiochemistry
Volume31
Issue number32
StatePublished - 1992
Externally publishedYes

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Epidermal Growth Factor
Epidermal Growth Factor Receptor
Cross-Linking Reagents
Size exclusion chromatography
Gel Chromatography
Esters
Labeling
Phosphotransferases
Carcinoma
Cells
Membranes
Derivatives
4-(fluorosulfonyl)benzoic acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "High-yied covalent attachment of epidermal growth factor to its receptor by kinetically controlled, stepwise affinity cross-linking",
abstract = "We report here the use of a stepwise affinity cross-linking technique in the specific covalent attachment of epidermal growth factor (EGF) to its receptor. A heterobifunctional cross-linking reagent, sulfo-TV-succinimidyl 4-(fluorosulfonyl)benzoate (SSFSB), which contains a rapidly reacting sulfo-N-succinimidyl active ester and a much more slowly reacting aromatic fluorosulfonyl moiety, was synthesized and characterized. Murine EGF (mEGF) was modified by the cross-linker to yield as the major product a derivative of mEGF having the (fluorosulfonyl)benzoyl moiety attached covalently at the amino terminus. SSFSB-modified, 125I-labeled mEGF was separated from unreacted SSFSB by size-exclusion chromatography and applied to shed membrane vesicles from A431 human carcinoma cells. Binding of derivatized 125I-mEGF to vesicles led to high yields (>60{\%}) of covalent linkage of 125I-mEGF to the EGF receptor, as determined by measurement of the fraction of specifically bound radiolabel which comigrated with the EGF receptor in NaDodSO4-polyacrylamide gels. The specificity of affinity cross-linking was evident in the negligible degree of labeling of species other than the EGF receptor and in the retention of EGF-stimulated receptor kinase activity after cross-linking.",
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N2 - We report here the use of a stepwise affinity cross-linking technique in the specific covalent attachment of epidermal growth factor (EGF) to its receptor. A heterobifunctional cross-linking reagent, sulfo-TV-succinimidyl 4-(fluorosulfonyl)benzoate (SSFSB), which contains a rapidly reacting sulfo-N-succinimidyl active ester and a much more slowly reacting aromatic fluorosulfonyl moiety, was synthesized and characterized. Murine EGF (mEGF) was modified by the cross-linker to yield as the major product a derivative of mEGF having the (fluorosulfonyl)benzoyl moiety attached covalently at the amino terminus. SSFSB-modified, 125I-labeled mEGF was separated from unreacted SSFSB by size-exclusion chromatography and applied to shed membrane vesicles from A431 human carcinoma cells. Binding of derivatized 125I-mEGF to vesicles led to high yields (>60%) of covalent linkage of 125I-mEGF to the EGF receptor, as determined by measurement of the fraction of specifically bound radiolabel which comigrated with the EGF receptor in NaDodSO4-polyacrylamide gels. The specificity of affinity cross-linking was evident in the negligible degree of labeling of species other than the EGF receptor and in the retention of EGF-stimulated receptor kinase activity after cross-linking.

AB - We report here the use of a stepwise affinity cross-linking technique in the specific covalent attachment of epidermal growth factor (EGF) to its receptor. A heterobifunctional cross-linking reagent, sulfo-TV-succinimidyl 4-(fluorosulfonyl)benzoate (SSFSB), which contains a rapidly reacting sulfo-N-succinimidyl active ester and a much more slowly reacting aromatic fluorosulfonyl moiety, was synthesized and characterized. Murine EGF (mEGF) was modified by the cross-linker to yield as the major product a derivative of mEGF having the (fluorosulfonyl)benzoyl moiety attached covalently at the amino terminus. SSFSB-modified, 125I-labeled mEGF was separated from unreacted SSFSB by size-exclusion chromatography and applied to shed membrane vesicles from A431 human carcinoma cells. Binding of derivatized 125I-mEGF to vesicles led to high yields (>60%) of covalent linkage of 125I-mEGF to the EGF receptor, as determined by measurement of the fraction of specifically bound radiolabel which comigrated with the EGF receptor in NaDodSO4-polyacrylamide gels. The specificity of affinity cross-linking was evident in the negligible degree of labeling of species other than the EGF receptor and in the retention of EGF-stimulated receptor kinase activity after cross-linking.

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