title: Cross-linked growth hormone dimers have enhanced biological activity.
abstract: In this study we have investigated the effect on the bioactivity of pituitary-derived human growth hormone (hGH) and recombinant bovine (b) GH after the addition of various concentrations of the water soluble cross-linking agent 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC; 6.25-100 mg/ml). The biological activity of resulting cross-linked reactions were determined by its ability to promote incorporation of 35SO4(2-) into costal cartilage of hypopituitary Snell dwarf mice in vivo. Administration of EDC-treated hGH solutions resulted in a significant enhancement of hormone activity in vivo compared with non-cross-linked samples. A similar significant enhancement of bGH activity in vivo was also observed when solutions containing recombinant bGH were cross-linked using EDC. For both hGH and bGH the degree of enhancement appears to be dose-dependent for the concentration of EDC (6.25-100 mg/ml for hGH; 6.25-50 mg/ml for bGH) present in the cross-linking reactions. SDS-PAGE analysis of EDC cross-linked solutions containing hGH and bGH spiked with 125I-hGH and 125I-bGH respectively revealed that dimeric GH was the primary cross-linked component. Increasing the concentration of EDC in cross-linking reactions resulted in increased formation of dimeric hGH and bGH. There was a significant correlation between the amount of GH dimer present and the increase in biological activity, suggesting that GH dimers were responsible for the enhanced biological activity. This was confirmed by the enhanced biological activity of a purified preparation of EDC cross-linked dimeric hGH. In conclusion, covalently cross-linked GH dimers reported here have enhanced bioactivity in vivo. However, since naturally occurring GH dimers are known to have reduced biological activity, this work suggests that the structure of EDC cross-linked GH dimers differs fundamentally from that of native dimeric hGH.
authors.affiliation: The Babraham Institute, Babraham, Cambridge, UK.
authors.forename: J W
authors.initials: JW
authors.lastname: Mockridge
authors.forename: R
authors.initials: R
authors.lastname: Aston
authors.forename: D J
authors.initials: DJ
authors.lastname: Morrell
authors.forename: A T
authors.initials: AT
authors.lastname: Holder
mesh_terms: D000818:Animals; D002356:Cartilage / Q000187:drug effects / Q000378:metabolism; D002417:Cattle; D003432:Cross-Linking Reagents; D019281:Dimerization; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004393:Dwarfism, Pituitary / Q000188:drug therapy*; D013006:Growth Hormone / Q000737:chemistry* / Q000494:pharmacology; D019382:Human Growth Hormone / Q000737:chemistry* / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D019382:Human Growth Hormone; D013006:Growth Hormone