EVIDENCE FOR 2 FOLDING DOMAINS IN GLYCOPROTEIN HORMONE ALPHA-SUBUNITS

We reconstituted ovine (o) LH alpha from its amino- and carboxyl-termi nal fragments obtained as follows. oLH alpha was nicked at Arg(46)-Ser (47) with Arg-C protease. Nicked oLH alpha disulfide bonds were broken by sulfitolysis, and its N-terminal peptide and C-terminal glycopepti de were separated by Sephacryl S-200 chromatography. Both fragments we re mixed, reduced, and reoxidized. Reoxidation products were chromatog raphed on Sephacryl S-200, and an alpha-monomer fraction was recovered . The putative nicked alpha-monomer fraction was reassociated with nat ive oLH beta, and the resulting oLH derivative was isolated by S-200 c hromatography with a reduced yield of 11% (intact subunits yield, 67% oLH). This preparation was 2.6% as active as oLH in a LH receptor bind ing assay. Two additional oLH derivatives were prepared Cleavage at al pha Arg(46)-Ser(47) alone, followed by reassociation with native oLH b eta, produced Arg-C-nicked oLH alpha:oLH beta (14% yield) that was 3.3 % as active as native oLH. Reduction-reoxidation of Arg-C-nicked oLH a lpha followed by reassociation with oLH beta produced reduced reoxidiz ed-Arg-C-nicked oLH alpha:oLH beta (11% yield) that was 1.8% as active as oLH. These results indicated that the nicked oLH alpha monomer had been reconstituted from its N- and C-terminal fragments.