Purification and receptor binding properties of complexes between lutropin and monovalent antibodies against its alpha subunit.
A complex between bovine lutropin (LH) and monovalent antibodies (Fab fragments) directed against its alpha subunit, which is common to the glycoprotein hormones, has been purified by gel filtration and chromatography on concanavalin A-Sepharose. The complex is heterogenous with respect to molecular size; 70--80% of the hormone is complexed with either two or three Fab fragments. The LH-Fab alpha complexes retain only about 13% receptor binding activity as compared to LH when measured in a radioligand receptor assay in which the radiolabeled ligand is human choriogonadotropin. (Use of the human hormone as labeled ligand permits direct measurement of competition between receptor and the bovine complex because the alpha portion of the human hormone does not cross react significantly with antibodies directed against bovine alpha subunits.) Complex formation does not lead to dissociation of the lutropin into its subunits, as shown with a homologous LH-beta immunoassay which distinguishes free beta subunit from intact LH. Complexing of LH with Fab-alpha fragments also causes little or no change in the affinity of the hormone's beta subunit for anti-LH-beta antibodies indicating that significant changes in beta subunit conformation did not occur. The data show that at least two well-separated antigenic regions on the alpha subunit are exposed to the surface in the intact hormone. They are also in agreement with the proposal that the loss of binding activity to receptor is due to steric effects rather than to changes in conformation or dissociation, and that there may be sites on the alpha subunit which interact directly with the receptor.
D000818:Animals; D001666:Binding Sites, Antibody; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification / Q000378:metabolism*; D007986:Luteinizing Hormone / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011863:Radioimmunoassay; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000378:metabolism*
D007140:Immunoglobulin Fab Fragments; D011956:Receptors, Cell Surface; D007986:Luteinizing Hormone