Determination of disulfide structure in agouti-related protein (AGRP) by stepwise reduction and alkylation.

Abstract	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.
Authors	E J Bures, J O Hui, Y Young, D T Chow, V Katta, M F Rohde, L Zeni, R D Rosenfeld, K L Stark, M Haniu
Journal	Biochemistry (Biochemistry) Vol. 37 Issue 35 Pg. 12172-7 (Sep 01 1998) ISSN: 0006-2960 [Print] United States
PMID	9724530 (Publication Type: Comparative Study, Journal Article)
Chemical References	AGRP protein, human Agatoxins Agouti Signaling Protein Agouti-Related Protein Disulfides Fluoresceins Intercellular Signaling Peptides and Proteins Peptide Fragments Proteins Spider Venoms omega-agatoxin-Aa4b Cysteine
Topics	Agatoxins Agouti Signaling Protein Agouti-Related Protein Alkylation Amino Acid Sequence Animals Cysteine (chemistry) Disulfides (chemistry, metabolism) Fluoresceins (metabolism) Humans Intercellular Signaling Peptides and Proteins Molecular Sequence Data Oxidation-Reduction Peptide Fragments (chemistry, isolation & purification, metabolism) Proteins (chemistry, isolation & purification, metabolism) Spider Venoms (chemistry)

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