Hormonotoxins: synthesis, characterization and bioefficacy of some defined disulfide linked conjugates of ovine LH with a ribosome inactivating protein, gelonin.
In order to synthesise a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, gelonin, a single chain RIP obtained from an Indian plant, Gelonium multiflorum of Euphorbeaceae family was covalently linked to oLH with the use of N-succinimidyl-3-(2-pyridyldithio)propionate, generating a linkage containing a disulfide bond and a amide bond. The hormonotoxins were separated according to their molecular weight (indirectly according to oLH:gelonin molar ratio) and a complete biochemical analysis was performed. The linkage occurred through the epsilon-NH2 group of alpha oLH as judged from RP-HPLC analysis. The conjugates were devoid of ingredients as determined by SDS-PAGE and RP-HPLC analysis. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity of oLH and gelonin to their antibodies. Hormonotoxins bind to the Leydig tumour cells via oLH part leaving gelonin free as judged by competitive displacement analysis. The hormonotoxin was internalized to the sufficient degree to effectively inhibit protein synthesis. The cytotoxicity of 1:1 molar ratio conjugate was relatively higher than that of others. The cytotoxicity of presently described more defined hormonotoxins exhibited higher receptor binding and cytotoxicity than the hormonotoxins reported earlier [Singh, et al., J Biol Chem, 264 (1989) 3089].
Singh
V
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Institute of Self-Organising Systems and Biophysics, North-Eastern Hill University, Shillong, India.
D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects; D003432:Cross-Linking Reagents; D066298:In Vitro Techniques; D007984:Leydig Cell Tumor / Q000188:drug therapy; D007986:Luteinizing Hormone / Q000494:pharmacology*; D010940:Plant Proteins / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011974:Receptors, LH / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects*; D012756:Sheep; D013388:Succinimides
D003432:Cross-Linking Reagents; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011974:Receptors, LH; D054789:Ribosome Inactivating Proteins, Type 1; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C025674:GEL protein, Gelonium multiflorum; D007986:Luteinizing Hormone