Formation and secretion of fragments of parathormone. Identification of cleavage sites.
Monolayer cultures of bovine parathyroid cells or fresh gland slices were incubated with radioactive amino acids in order to study the formation and metabolism of parathormone (PTH). PTH, secretory protein I, and COOH-terminal fragments of PTH were all released into media within 30 min, most strongly in the first hour after synthesis. Peptides in tissue, cells, and media were separated using reverse-phase high performance liquid chromatography. In eluates of media, six radioactive peaks were prominent. The first four and the sixth were immunoreactive in a COOH-terminal specific PTH radioimmunoassay, but only the sixth was reactive in an NH2-terminal specific assay. Under conditions where recovery of PTH(1-34) was quantitative, gel filtration of media was used to show that no NH2-terminal fragments of PTH were secreted. Sequence analyses of secreted COOH-terminal peptides indicated that the NH2 termini of the first three peaks corresponded to residues 43, 37, and 34 of PTH. The fourth peak contained a mixture of two peptides with NH2 termini at residues 24 and 28 of PTH. The fifth could not be identified; the sixth was PTH. Cleavages at the 23-24 bond of PTH occurred within minutes of the formation of PTH itself, and the other peptides were formed more slowly. Mandatory cleavage of PTH at the 23-24 peptide bond would destroy the biological activity of the hormone on kidney and bone, a situation consistent with the possibility that intracellular PTH metabolism participates in secretory regulation. The results showed that different peptides were generated in parathyroid cells than were previously shown to be produced by cathepsin B or D. The results suggest that the proteolytic pathway which results in the secretion of PTH fragments is nonlysosomal in nature.
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D053379:Chromogranin A; D002864:Chromogranins; D006056:Golgi Apparatus / Q000378:metabolism; D010280:Parathyroid Glands / Q000187:drug effects / Q000378:metabolism; D010281:Parathyroid Hormone / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000378:metabolism; D012636:Secretory Rate / Q000187:drug effects
D002135:Calcium-Binding Proteins; D053379:Chromogranin A; D002864:Chromogranins; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D002118:Calcium