Purification and characterization of a fetal somatomedin from the sheep: similarity to insulin-like growth factor II.
A high efficiency procedure for the purification to homogeneity of an ovine fetal somatomedin is described. Fetal sheep serum was used as the source material, and activity was followed throughout purification by an insulin-like growth factor (IGF) II RRA. IGF-II-like activity was initially enriched through binding at acid pH to a column of SP-Sephadex C-25 and elution with a neutral pH high-salt buffer. Further chromatography on SP-Sephadex resulted in a preparation containing less than 0.1% of the original serum protein content, but retaining much of the IGF-II-like activity. This enriched fetal IGF preparation was then purified to homogeneity using reverse phase HPLC. However, chromatography on an HPLC gel filtration column was found to be essential to ensure the stability of the purified peptide during storage, although this procedure did not result in any apparent increase in purification. The final yield of purified ovine fetal IGF was 80 micrograms from 400 ml fetal sheep serum. Only one polypeptide chain was detected during amino-terminal sequencing of the fetal somatomedin, and the preparation gave a single band on both gel electrophoresis and isoelectric focusing, indicating that the peptide was essentially pure. The sequence (eight amino acid residues) was identical to the equivalent sequence in IGF-II from human, rat, and bovine sources. In additional, amino acid analysis of the ovine fetal IGF showed close similarity to the amino acid content of IGF-II from other species. The mol wt of the purified peptide, estimated by HPLC gel filtration, was approximately 7000, close to that of previously purified somatomedins, and the isoelectric point, obtained by chromatofocusing, was around pH 6.8. Thus, the purified ovine fetal somatomedin appears to be similar to IGF-II from other species, and may be the ovine homolog of human IGF-II.
Hey
A W
AW
Department of Physiology, Monash University, Clayton, Victoria, Australia.
Browne
C A
CA
Thorburn
G D
GD
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005312:Fetal Blood / Q000032:analysis*; D007335:Insulin-Like Growth Factor II / Q000097:blood*; D008970:Molecular Weight; D011863:Radioimmunoassay; D012756:Sheep; D013002:Somatomedins / Q000097:blood*
D000596:Amino Acids; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II