Metabolism and biological activity of proparathyroid hormone and synthetic analogues in renal cortical membranes.
The metabolism of natural and synthetic analogues of bovine proparathyroid hormone (Pro-bPTH) and the biological activity of the synthetic fragments were evaluated in an in vitro assay employing renal cortical membranes (adenylyl cyclase bioassay). Apparent biological activity of the prohormone analogue was correlated with the cleavage of prohormone to hormone by the membranes. Analysis by electrophoresis on polyacrylamide gels and by ion-exchange chromatography on carboxymethyl cellulose (CMC) of the synthetic analogue Pro-bPTH-(-6 leads to +34) labelled with 125I, after incubation with renal cortical membranes, revealed conversion of the analogue to a fragment co-migrating or co-eluting with bPTH (1-34). Similar electrophoretic analyses using biosynthetic Pro-bPTH-(-6 leads to +84) internally labelled with [3H] leucine revealed degradation of Pro-bPTH-(-6 leads to +84) to smaller fragments. Proteolysis of both native prohormone and prohormone analogue was markedly reduced in incubations performed in the presence of benzamidine, a competitive inhibitor of trypsin and trypsin-like enzymes. Inclusion of benzamidine in incubations with purified renal cortical membranes from rat or dog in the in vitro renal adenylyl cyclase bioassay resulted in a ten-fold lowering of the potency of the fragment Pro-bPTH-(-6 leads to +34) relative to that of bPTH-(1-34); the potency of Pro-bPTH-(-6 leads to +34) was reduced from 5.4-3.6% to 0.5-0.3%, on a molar basis, of the potency of bPTH-(1-34). There was no effect of benzamidine on the intrinsic activity of bPTH-(1-34). These studies indicate that most if not all of the apparent biological activity of Pro-bPTH-(-6 leads to +34), at least in vitro, is dependent upon prior enzymic conversion to bPTH-(1-34).
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000937:Antigen-Antibody Reactions; D001550:Benzamidines / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006728:Hormones / Q000378:metabolism; D007672:Kidney Cortex / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010281:Parathyroid Hormone / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D011498:Protein Precursors / Q000502:physiology*; D051381:Rats
D001550:Benzamidines; D006728:Hormones; D010281:Parathyroid Hormone; D011498:Protein Precursors; D000262:Adenylyl Cyclases