Structural analysis of recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor.
The recombinant human carboxy-terminal-truncated macrophage colony-stimulating factor ([3-153]M-CSF) consists of 302 amino acid residues and has a molecular mass of about 32 kDa, as estimated by SDS-PAGE. Two covalently linked subunits constitute a bioactive homodimer. The structure of the purified protein, expressed in Escherichia coli and refolded from inclusion bodies, was studied. The amino acid sequence was determined by automated Edman degradation of fragments obtained from degradation with CNBr and iodosobenzoic acid as well as by digestion with Glu-C endopeptidase of reduced and alkylated M-CSF. The absence of free thiol groups in the molecule was confirmed with Ellman reagent, which indicated the presence of seven disulfide linkages per homodimer. Sequence analysis of cystine-containing peptides, identified by comparing the peptide maps from unmodified and performic acid-oxidized pepsin digests, gave the following results. (1) Six out of seven disulfide linkages were formed between Cys 7 and Cys 90, Cys 48 and Cys 139, and Cys 102 and Cys 146 at each pair of positions as either intra- or inter-chain disulfides. (2) The remaining disulfide linkage linked Cys 31 of one subunit to Cys 31 of the second subunit of M-CSF. Based on our findings, a two-dimensional model is proposed in which the possible covalent linkage is suggested between the two subunits of the bioactive [3-153]M-CSF molecule.
Yamanishi
K
K
Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Tokushima.
Randawa
Z I
ZI
Brown
D
D
Masui
Y
Y
Yasuda
S
S
Takahashi
M
M
Adachi
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis
D000596:Amino Acids; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D003545:Cysteine