Assignment of disulfide bond location in prothoracicotropic hormone of the silkworm, Bombyx mori: a homodimeric peptide.
The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion. The partial reduction and S-alkylation broke the interchain disulfide bond but did not affect the intrachain disulfide bonds, generating monomeric PTTH whose intrachain disulfide bonds were kept intact. This monomeric PTTH has about one-half the biological activity of intact PTTH. Sequence analysis of the fragments generated by lysyl endopeptidase digestion of this monomeric PTTH after complete reduction and S-alkylation by another S-alkylating reagent showed that only the Cys15 residue was reduced and S-alkylated by the foregoing partial reduction, indicating that this residue formed the interchain disulfide bond. The other disulfide bonds which formed intrachain bridgings were determined by sequence and mass analyses of the fragments generated by two successive enzyme digestions of the monomeric PTTH. In conclusion, the disulfide bond location of PTTH was assigned to Cys15-Cys15' as an interchain disulfide linkage and Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98 as intrachain disulfide linkages.
Ishibashi
J
J
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Kataoka
H
H
Isogai
A
A
Kawakami
A
A
Saegusa
H
H
Yagi
Y
Y
Mizoguchi
A
A
Ishizaki
H
H
Suzuki
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000737:chemistry*; D004220:Disulfides / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D007301:Insect Hormones / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry
D004220:Disulfides; D007301:Insect Hormones; D011994:Recombinant Proteins; C009089:prothoracicotropic hormone