STUDIES OF HUMAN PITUITARY LH CONTAINING INTERNALLY CLEAVED BETA-SUBUNIT

We have investigated the origin of the internal peptide bond cleavage found in the beta-subunit of a proportion of pituitary human LH (hLH) molecules, as well as the effects of this cleavage (or nick) on the interaction between alpha and beta-subunits and on binding of hLH to its receptor. The content of cleaved beta-subunit, assessed by the intensity of staining of an approximately 10 kDa component on sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced hLH, was variable in batches of hLH prepared from pooled acetone-preserved human pituitary glands. There was evidence of a similar cleavage in purified hTSH, but not in the purified hFSH or human chorionic gonadotrophin examined. Although intact hLH was relatively resistant to cleavage in solution, urea dissociation of hormone followed by dialysis resulted in an increased content of nicked beta-subunit, which was largely prevented by incorporation of the proteolytic enzyme inhibitor phenylmethylsulphonyl fluoride (PMSF) and the metal-chelating agent EDTA. Hormone that was virtually free of nicked beta-subunit was obtained by urea dissociation of hLH subunits in the presence of PMSF and EDTA followed by dialysis to remove urea, reassociation of subunits at 37-degrees-C (pH 7) and purification of reassociated hLH dimer by gel filtration on Sephadex G-100 in the presence of 1-anilinonaphthalene-8-sulphonic acid (ANS). When hLH was incubated at 37-degrees-C at pH 5.5 or pH 9.5 in the absence of enzyme inhibitors, some cleaved beta-subunit was found in the hLH-ANS dimer form of the hormone, but most of the nicked subunit appeared in fractions of lower molecular weight and with low receptor-binding activity. Fractions isolated as hLH-ANS dimer had receptor-binding activities which were negatively correlated with their content of cleaved beta-subunit, the most active fraction (21 000 i.u./mg) being virtually free of this component. Our studies suggest (1) that the cleavage is caused by proteolytic enzyme activity present in human pituitary tissue and in purified preparations of hLH, (2) that free hLH-beta-subunit is cleaved far more readily than when it is combined with alpha-subunit in the intact hormone, (3) that the presence of the cleavage in free hLH-beta prevents refolding of this subunit to the correct conformation that permits its combination with alpha-subunit to regenerate native hormone, and (4) that the site of cleavage is likely to be at a peptide bond located on the surface of the intact hLH molecule.