UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Isolation and characterization of salmon ultimobranchial calcitonin O'Dor, Ronald Keith


Although the first preparations of the hypocalcemic hormone, calcitonin (ct), were extracted from rat thyroid glands, histological evidence showed that the "c" cells which produce the hormone are not restricted to this gland, but also occur in the parathyroid and thymus tissues of mammals. These cells arise embryologically from the last branchial pouch and in non-mammals they form a separate ultimobranchial gland which also contains hypocalcemic activity. The work described in this thesis provides evidence that this activity results from polypeptides structurally similar to those isolateo from mammalian thyroid tissues and.explores the relationship between the structural and functional differences of the two types of ct. A survey of four mammalian thyroid tissues (human, bovine, porcine and murine) ano four non-mammalian ultimo-branchial tissues (turkey, chicken, salmon and dogfish) demonstrated that these tissues contained hypocalcemic polypeptides with molecular weights of about 4000 as determined by gel filtration. When extracts were prepared using an organic solvent mixture developed for the thyroid ct' s the ultimobranchial tissues yielded more hypocalcemic activity on a fresh weight basis and the final product had a higher specific activity. Salmon ultimobranchal tissue was collected on a large scale and extracted to provide material for chemical characterization. A series of three gel filtration stages on sephaoex g-50 alternating with two ion-exchange chromatography stages on se-sephadex c-25 at two ph's provided a 300,000 fold purification and yielded 15 mg of pure salmon ct. amino acid analysis and partial characterization of tryptic peptides indicated that the ultimobranchial hormone was a 32 amino acio polypeptide with a disulfide bridge at the c-terminus. Although these features are also common to all the mammalian ct's, there are a number of unique features in the salmon ct structure. These structural differences were also reflected in the biological activity of the hormone. Salmon ct was nearly 50 times more active than human ct in the standard bioassay and the response to the salmon hormone was prolonged. Tests in plasma both in vivo and in vitro indicated that salmon ct was much more stable than the 'thyroid ct' s suggesting a possible reason for its greater potency. A survey of cohn fractions from human plasma showed that fractions iii-o, iv-1 iv-4 contained enzymes capable of rapidly degrading porcine ct. in further studies on fraction iv—1 a selective "calci-toninase" was purified by chromatography on deae-sephadex, sephadex g-200 and cm-sephadex. this enzyme rapidly inactivated porcine ct, but had no significant effect on salmon or human ct. synthetic porcine ct was digested with the enzyme and the resultant peptides were isolateo and identified. The nature of these peptides indicated that the enzyme was a peptidase with a specificity for the carboxyl side of arginine residues. similar digestions of pure native salmon ct produced no peptides providing at least a partial explanation for the greater stability of this hormone. These experiments also showed that the enzyme would not split at all arginine residues and would not cleave bonds associated with other basic residues. The data indicated that the enzyme had a molecular weight of about 30,000 and probably was derived from a precursor with a molecular weight of about 100,000. Tests of the action of the enzyme on fibrinogen showed that it was not thrombin. comparison of available data with that for other plasma enzymes indicated similarities to the kallikrein family of enzymes, but the "calcitoninase" does not appear to be identical with any of the well studies members of this group.

Item Media

Item Citations and Data


For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.