Partial purification and characterization of somatomedin from sheep serum.
A rapid, high yield preparative technique for the isolation of sheep somatomedin is reported. Purification of biologically active somatomedin from the 60% ammonium sulfate precipitate of sheep serum was accomplished using three gentle fractionation steps. Biological activity during purification was monitored using the rat adipocyte nonsuppressible insulin-like activity (NSILA) assay. A stepwise pH elution (pH 2.85, 3.5, 4.5, and 6.0) from SP-Sephadex resulted in the elimination of more than 99% of the serum proteins and a 500-fold enhancement of biological activity. The active fraction eluted at pH 6.0 and was further fractionated on Sephadex G-50 (fine) chromatography at pH 2.85. This resulted in about a 10,000-fold enhancement of activity over serum activity. The most active fractions from Sephadex chromatography were further separated on reverse phase HPLC in 0.1% trifluoroacetic acid using a linear gradient of 24-60% acetonitrile. The biological activity of the final preparation was enhanced 61,000- to 182,000-fold over that of serum (mean, 93,000-fold) when assayed in the NSILA assay. Protein yield was estimated to be 467 micrograms/liter serum. In addition to the NSILA activity, the final preparation demonstrated dose-dependent sulfation factor activity in the embryonic chick pelvic leaflet bioassay. Sheep somatomedin was active at physiological levels in both bioassays. Analysis of the somatomedin preparation by sodium dodecyl sulfate-electrophoresis at pH 8.8 showed that it was homogeneous by this criterion. The activity eluted from Sephadex G-50 was estimated to have a molecular size of 6900. Two Coomassie blue-stained bands were present in the final sheep somatomedin preparation after polyacrylamide gel electrophoresis at pH 3.2. Our purification process is a rapid, high yield technique which yields a polypeptide fraction enriched in NSILA and somatomedin-like activity. The molecular size and biological activity in the NSILA and sulfation factor assays suggest that our sheep NSILA is analogous to somatomedins purified from other species of animals.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012756:Sheep / Q000378:metabolism*; D013002:Somatomedins / Q000097:blood* / Q000302:isolation & purification / Q000032:analysis
D013002:Somatomedins