Determination of disulfide structure in agouti-related protein (AGRP) by stepwise reduction and alkylation.
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.
Amgen, Inc., Thousand Oaks, California 91320, USA.
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Bures
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Hui
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Young
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Chow
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Katta
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Rohde
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Zeni
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Rosenfeld
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Stark
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Haniu
D060848:Agatoxins; D054366:Agouti Signaling Protein; D054369:Agouti-Related Protein; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013111:Spider Venoms / Q000737:chemistry
C515198:AGRP protein, human; D060848:Agatoxins; D054366:Agouti Signaling Protein; D054369:Agouti-Related Protein; D004220:Disulfides; D005452:Fluoresceins; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011506:Proteins; D013111:Spider Venoms; C083855:omega-agatoxin-Aa4b; D003545:Cysteine