Conversion of proparathyroid hormone to parathyroid hormone: studies in vitro with trypsin

Abstract

The conversion of proparathyroid hormone to parathyroid hormone (PTH) was studied in vitro employing pancreatic trypsin as a prototype converting enzyme. Digestion of intact radiolabeled bovine prohormone with trypsin (0.1%) (wt/wt) resulted in release of a peptide comigrating with intact hormone marker in systems resolving on the basis of charge (urea polyacrylamide gels, pH 4.4) and size (sodium dodecyl sulfate-urea polyacrylamide gels, pH 7.2). Tryptic digestions of a synthetic analogue of bovine prohormone ProPTH-(-6 to +34), consisting of the prohormone hexapeptide covalently bonded to the NH2 terminus of the active fragment of the hormone, released in high yield the hexapeptide and the intact active hormone fragment before any other smaller fragments. Analyses of digestions were by TLC and amino acid analysis of digestion products; comparison of the biological activity of the prohormone substrate and the products of digestion; and peptide endgroup analysis by the Edman method during progressive tryptic hydrolysis over 22 h. The latter experiments demonstrated cleavage of more than 75% of the hexapeptide-hormone peptide bond before cleavage of other trypsin-sensitive sites within the molecule. The specificity of cleavage at the hexapeptide-hormone bond in the process of intracellular hydrolysis of proparathyroid hormone resides primarily in the sequence and/or conformation of the precursor molecule. Since conversion of prohormone to hormone can be efficiently accomplished by pancreatic trypsin in vitro, there is no need to postulate the existence of an intracellular converting enzyme within the parathyroid cell that possesses unique hydrolytic specificity.