Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Location of specifically modified methionine residues.
Bovine lutropin (luteinizing hormone) was carboxymethylated at pH3.0 for 12 h at 37 degrees C with iodoacetic acid for specific modification of methionine residues. To facilitate the location of preferentially modified methionine residues, iodoE114C]acetic acid was added as tracer. The alpha and beta subunits of bovine lutropin were carboxymethylated with a 2- or 5-fold molar excess of iodoacetic acid either in the presence or absence of their counterpart subunits. The modified subunits were separated and isolated by counter-current distribution followed by gel filtration on Sephadex G-100. To locate the modified methiones, the isolated alpha or beta chain was reduced. S-carboxymethylated and subjected to tryptic hydrolysis. The tryptic peptides were fractionated by gel filtration on Bio-Gel P-10. From analyses of the purified 14C-labelled tryptic peptides, it was observed that methionine-8 and -33 in bovine lutropin alpha chain and methionine-52 in the beta chain were preferentially modified. Similar results were obtained when isolated alpha and beta subunits were individually carboxymethylated in the absence of their counterpart subunit under identical conditions. The fact that a recombinant of native human lutropin alpha chain, in which a valine residue is present in the position corresponding to methionine-8 of bovine lutropin alpha chain, and carboxymethylated bovine lutropin beta chain regenerated a substantial amount of receptor-site-binding activity indicated that methionine-8 in bovine alpha chain was biologically not essential. These studies showed clearly that both methionine-33 in the alpha chain and methionine-52 in the beta subunit were involved for optimum binding between bovine lutropin and its receptors for expression of hormonal activity.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D007461:Iodoacetates; D007986:Luteinizing Hormone / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D008745:Methylation; D014357:Trypsin
D000596:Amino Acids; D007461:Iodoacetates; D007986:Luteinizing Hormone; D008715:Methionine; D014357:Trypsin