Evidence for two folding domains in glycoprotein hormone alpha-subunits.
We reconstituted ovine (o) LH alpha from its amino- and carboxyl-terminal fragments obtained as follows. oLH alpha was nicked at Arg46-Ser47 with Arg-C protease. Nicked oLH alpha disulfide bonds were broken by sulfitolysis, and its N-terminal peptide and C-terminal glycopeptide were separated by Sephacryl S-200 chromatography. Both fragments were mixed, reduced, and reoxidized. Reoxidation products were chromatographed on Sephacryl S-200, and an alpha-monomer fraction was recovered. The putative nicked alpha-monomer fraction was reassociated with native oLH beta, and the resulting oLH derivative was isolated by S-200 chromatography with a reduced yield of 11% (intact subunits yield, 67% oLH). This preparation was 2.6% as active as oLH in a LH receptor binding assay. Two additional oLH derivatives were prepared. Cleavage at alpha Arg46-Ser47 alone, followed by reassociation with native oLH beta, 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 alpha followed by reassociation with oLH beta produced reduced reoxidized-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.
Bousfield
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GR
Department of Biological Sciences, Wichita State University, Kansas 67260.
Ward
D N
DN
D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006736:Horses; D006801:Humans; D007700:Kinetics; D007986:Luteinizing Hormone / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011974:Receptors, LH / Q000378:metabolism; D017385:Sequence Homology; D012697:Serine Endopeptidases / Q000378:metabolism; D012756:Sheep
D004220:Disulfides; D015292:Glycoprotein Hormones, alpha Subunit; D010446:Peptide Fragments; D011974:Receptors, LH; D007986:Luteinizing Hormone; C044499:submandibular proteinase A; D012697:Serine Endopeptidases