Binding protein, radioreceptor and biological activities of recombinant methionyl insulin-like growth factor-I variants.
Reversed-phase chromatography (RPC) was used to resolve two variants of recombinant amino terminal methionyl residue (N-Met) insulin-like growth factor-I (IGF-I) with the same amino acid constitution but different disulphide linkages. Following radioiodination, equilibration with plasma and size exclusion chromatography at neutral pH the major form on RPC (approximate abundance 60%) demonstrated greater than 80% binding to 150 kDa and 40-50 kDa IGF binding proteins. This peptide has the RPC elution characteristics and disulphide assignment (Cys6-Cys48, Cys18-Cys61, Cys47-Cys52) of authentic with mismatched disulphides (Cys6-Cys47, Cys18-Cys61, Cys48-Cys52; N-Met IGF-I peak 1 peptide) demonstrated less than 15% binding under similar conditions. Potency of the peptides was investigated in competitive IGF-I plasma binding protein and L6 myoblast radioreceptor assays. The peak 2 peptide proved equipotent to purified ovine plasma IGF-I in each system but by contrast the peak 1 peptide was 40-fold and 200-fold less potent in the binding protein and radioreceptor assays respectively. Biological potency was examined in a non-competitive assay based on incorporation of [3H]leucine into confluent cultures of L6 myoblasts. In this system the N-Met IGF-I peak 1 peptide proved 15-fold less potent than the peak 2 peptide with correct disulphide linkages. Refolding variants may prove useful in establishing structure/function relationships for IGF-I.
Hodgkinson
S C
SC
Ruakura Agricultural Centre, Ministry of Agriculture and Fisheries, Hamilton, New Zealand.
Napier
J R
JR
Davis
S R
SR
Patel
B
B
Gluckman
P D
PD
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001681:Biological Assay; D001798:Blood Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology / Q000031:analogs & derivatives; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011869:Radioligand Assay; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology
D000596:Amino Acids; D001798:Blood Proteins; D002352:Carrier Proteins; D018969:Insulin-Like Growth Factor Binding Proteins; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; C061965:insulin-like growth factor I, N-methionyl-; D007334:Insulin-Like Growth Factor I