Covalent cross-linking of the bovine somatotropin dimer. Effects on growth-promoting, receptor-binding and immunological activities and preliminary characterization of the self-association.
Bovine somatotropin, at pH 8.5 in 0.02 M-Bicine [NN-bis-(2-hydroxyethyl)glycine]/0.09M-NaCl, showed by frontal analysis the characteristics of a rapid monomer-dimer equilibrium whose dissociation constant was estimated to be 6.6 X 10(-6)M. Reaction of the hormone with dimethyl suberimidate lead to covalent cross-linking of the dimeric species. Under the conditions chosen (0.4 mg of bifunctional imidate and 1 mg of protein/ml at room temperature for 1 h) the cross-linked dimers accounted for 26% of the total protein, and these were isolated by molecular sieving in 0.29M-NH3/0.12M-NaCl. Covalent stabilization greatly diminished the growth-promoting activity and the ability to interact with somatogenic sites in both rat liver in vivo and rabbit liver microsomal fractions. Evidence indicating a non-critical role for amino groups involved in the covalent cross-linking was provided by a nearly equivalent derivative obtained after reaction with 3,3'-dithiobispropionimidate, which had substantial hormonal activity upon cleavage of the disulphide links. Conversely, immunological reactivity as demonstrated by radioimmunoassay was not affected by cross-linking. Details of the least-squares procedure employed to evaluate the self-association equilibrium constant has been deposited as Supplement SUP 50115 (7 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem.J. (1981) 193,5.
D000818:Animals; D001835:Body Weight / Q000187:drug effects; D002417:Cattle; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D005260:Female; D013006:Growth Hormone / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007096:Imidoesters / Q000494:pharmacology; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D011817:Rabbits; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000378:metabolism; D011986:Receptors, Somatotropin
D003432:Cross-Linking Reagents; D007096:Imidoesters; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D011986:Receptors, Somatotropin; C024244:dithiobispropionimidate; D004120:Dimethyl Suberimidate; D013006:Growth Hormone