Studies of the biological and immunological properties of parathyroid hormone, labeled selectively on the methionine residues by [3H]methyl exchange to high specific activity.
A technique is described for labeling bovine parathyroid hormone (bPTH) with tritium by [3Hmethyl exchange. The methionine residues were first methylated with [3H]methyl iodide at pH 4, and the reaction products were separated by cation exchange chromatography. The major peak consisted to hormone in which both methionines were converted to [3H]methyl methionine sulfonium iodide (3H-methylated bPTH). This product was then demethylated with 2-mercaptoethanol (6 M) at pH 8.6 to regenerate the hormone in an unmodified but tritiated form ([3H]bPTH), with a specific activity of 1.7 Ci/mmol. High pressure liquid chromatographic analysis showed that 96% of the radioactivity was incorporated into the methionine residues. There was no evidence of any alteration in the primary structure, as [3H]bPTH was found to run in the same position as unlabeled bPTH on cation exchange chromatography and disc gel electrophoresis and to have an identical absorption spectrum in the 240- to 330-nm range. Moreover, [3H]bPTH had full biological activity, as measured by an in vitro bioassay based on activation of rat renal cortical adenylate cyclase, although 3H-methylated bPTH was almost completely inactive. Similarly, while 3H-methylated bPTH had reduced potency in a RIA specific for antigenic sites in the 1--34 region of the sequence, [3H]bPTH was found to have full activity. The preparation of labeled bPTH was repeated using [14C]methyl iodide, with similar results, although [14C]bPTH was found to have somewhat reduced immunological and biological activities. While [3H]bPTH had a lower specific activity than can be obtained by various other techniques for incorporating tritium or 125I into peptides, biosynthetic labeling is at present the only alternative method for preparing biologically active, labeled bPTH without altering the primary structure. By comparison with this technique, the present method gave a product of a much higher specific activity which was labeled specifically in the biologically essential amino-terminal region. The same simple chemical procedures are clearly of wide potential application to the preparation of other labeled peptides.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D001681:Biological Assay; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D007553:Isotope Labeling / Q000379:methods; D008715:Methionine; D008745:Methylation; D010281:Parathyroid Hormone / Q000494:pharmacology; D011863:Radioimmunoassay; D051381:Rats; D014316:Tritium
D010281:Parathyroid Hormone; D014316:Tritium; D008715:Methionine; D000262:Adenylyl Cyclases