Disulfide linked alpha oLH-gelonin conjugate failed to recombine with beta oLH subunit to generate bioeffective hormonotoxin.
Since, linking of ovine luteinizing hormone (oLH) to ribosome inactivating protein gelonin (in oLH-gelonin conjugate) occur via the alpha-subunit, alpha oLH, an attempt has been made to develop a 'universal' hormonotoxin for selective targeting to specific cells in the gonads. Four different molar ratios of oLH and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) were used to activate the epsilon amino (phi-NH2) groups of alpha oLH. The alpha oLH-SPDP derivatives recombine to native beta subunit of oLH (beta oLH) and the purified recombinants retained substantial receptor binding, steroidogenic activity and immunoreactivity to native oLH. The disulfide linked alpha oLH-S-S-gelonin conjugates prepared by SPDP method were purified by gel filtration chromatography and analysed by reverse-phase high performance liquid chromatography (RP-HPLC). In order to obtain specificity and bioeffectivity, the alpha oLH-S-S-gelonin conjugates were allowed to recombine to native beta oLH and the recombination mixture was further purified by gel-filtration chromatography. The RP-HPLC analysis of these recombinants indicated that alpha oLH-S-S-gelonin did not recombine to beta oLH. The failure of recombination may be due to the reasons. (i) The site of epsilon-NH2 activation by SPDP may be different in the alpha oLH than the native oLH. (ii) The activation site may be in close proximity to the annealing site which facilitates the recombination of beta-subunit but failed to reassociate to alpha oLH-S-S-gelonin conjugate.(ABSTRACT TRUNCATED AT 250 WORDS)
Singh
V
V
Institute of Self Organising Systems and Biophysics, North-Eastern Hill University, Shillong, Meghalaya, India.
Curtiss
R
R
D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004229:Dithiothreitol; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry*; D007986:Luteinizing Hormone / Q000737:chemistry*; D008297:Male; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry* / Q000633:toxicity; D055503:Protein Multimerization; D011869:Radioligand Assay; D051381:Rats; D054789:Ribosome Inactivating Proteins, Type 1; D012756:Sheep; D013388:Succinimides; D013737:Testis / Q000378:metabolism
D003432:Cross-Linking Reagents; D015292:Glycoprotein Hormones, alpha Subunit; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D013388:Succinimides; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C025674:GEL protein, Gelonium multiflorum; D007986:Luteinizing Hormone; D004229:Dithiothreitol