Modification of ovine luteinizing hormone subunits with SMPT and its effect on subunit recombination, immunological activity, receptor binding and steroidogenic activity.
The increasing use of heterobifunctional cross-linking agents in the design of defined conjugates for selective targeting and inducing immune response has prompted us to study the role of epsilon-NH2 group modification of oLH subunits, their recombination and effect on immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of alpha oLH and beta oLH subunits were separately modified by using SMPT. The alpha oLH-SMPT modified derivatives hybridize to beta oLH. Similarly, the beta oLH-SMPT derivatives recombined with alpha oLH. The recombination was judged by gel filtration chromatography and RP-HPLC analysis. The sequential modification of subunits led to progressive reduction in immunoreactivity and receptor binding activity. The modification of six or more epsilon-NH2 groups in alpha oLH although recombine fully with native beta oLH but failed to react to anti-oLH antibody. Moreover, the steroidogenic activity was also abolished. Introduction upto four SMPT groups in alpha oLH compromised immunological and biological activities but further addition of two or more SMPT groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two amino groups in the receptor binding and steroidogenic activity. The present investigation clearly demonstrate that only 1:2-3 molar ratio of oLH subunits:SMPT could generate the site(s) in the subunits of the oLH that retained reasonable immunological, receptor binding and biological activity of the hormone. Therefore, this molar ratio may be used in future for the design and synthesis of bioeffective hormonotoxins.
Singh
V
V
Hormone Biochemistry Laboratory, North-Eastern Hill University, Meghalaya, India. vsingh@nehus.ren.nic.in
Singh
R C
RC
D000818:Animals; D003432:Cross-Linking Reagents / Q000737:chemistry*; D007986:Luteinizing Hormone / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011869:Radioligand Assay; D011974:Receptors, LH / Q000378:metabolism*; D012756:Sheep; D013388:Succinimides / Q000737:chemistry*
D003432:Cross-Linking Reagents; D011974:Receptors, LH; D013388:Succinimides; C058151:4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene; D007986:Luteinizing Hormone