Determination of disulfide structure in agouti-related protein (AGRP) by stepwise reduction and alkylation
| Autor: | Kevin Lee Stark, Yunjen Young, John O. Hui, David T. Chow, Edward J. Bures, Michael F. Rohde, Lisa Zeni, Mitsuru Haniu, Robert Rosenfeld, Vishwanatham Katta |
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| Rok vydání: | 1998 |
| Předmět: |
Tris
Alkylation Sequence analysis Molecular Sequence Data Spider Venoms Agatoxins Biochemistry law.invention chemistry.chemical_compound law Animals Humans Agouti-Related Protein Amino Acid Sequence Cysteine Disulfides Peptide sequence Acidic Region digestive oral and skin physiology Proteins Fluoresceins Peptide Fragments chemistry Recombinant DNA Agouti Signaling Protein Intercellular Signaling Peptides and Proteins Oxidation-Reduction Phosphine |
| Zdroj: | Biochemistry. 37(35) |
| ISSN: | 0006-2960 |
| Popis: | The agouti-related protein gene (Agrp) plays an important role in body weight regulation. The mature human protein is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The disulfide structure of recombinant human AGRP was determined by chemical methods using partial reduction with tris(2-carboxyethyl)phosphine under acidic conditions, followed by direct alkylation with N-ethylmaleimide or fluorescein-5-maleimide. Partial reduction and alkylation provided several forms of AGRP that were modified in a stepwise fashion. The resulting proteins were characterized by peptide mapping, sequence analysis, and mass spectrometry, showing that AGRP contained a highly reducible disulfide bond, C85-C109, followed by less reactive ones, C90-C97, C74-C88, C67-C82, and C81-C99, respectively. The chemically defined disulfide connectivity of the recombinant human AGRP was homologous to that of omega-agatoxin IVB except for an additional disulfide bond, C85-C109. |
| Databáze: | OpenAIRE |
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