Stability of the Hypocalcemic Activity of Porcine Thyrocalcitonin

Abstract

Porcine thyrocalcitonin was modified by enzymatic and chemical treatment for the purpose of studying the interrelationships between structure and hypocalcemic activity. Enzymatic treatment with trypsin, chymotrypsin, pepsin and polyphenyl oxidase led to rapid and complete inactivation of the hypocalcemic activity. We conclude from these experiments that thyrocalcitonin either is a simple polypeptide or contains a peptide moiety which is absolutely essential for hypocalcemic activity. Incubation of thyrocalcitonin in dilute HC1 or neutral Tris buffer at elevated temperature was accompanied by gradual loss of activity. Inactivation was also seen after treatment with hydrogen peroxide, photooxidation and treatment with n-bromosuccinimide, results demonstrating that there is at least one site in the thyrocalcitonin molecule that is sensitive to oxidation. The hypocalcemic activity which was lost during incubation with hydrogen peroxide could not be recovered by treatment of the oxidized products with cysteine, 2-mercaptoethanol or dithiothreitol. There were no detectable changes in hypocalcemic activity either after enzymatic treatment with carboxypeptidase A, leucine aminopeptidase and neuraminidase, or after chemical treatment with 2-OH-5-nitrobenzyl bromide, N-acetylimidazole, p-OHmercuribenzoate, iodoacetate and cyanogen bromide. Because thyrocalcitonin has not yet been purified, the sites of the modifications produced by various treatments cannot be shown. Results of experiments in which no loss of hypocalcemic activity was detected must be viewed with caution since it was not possible to show that amino acid residues in thyrocalcitonin were modified. However, these data may help to explain losses of hypocalcemic activity which occur during handling and to suggest new approaches to purification and characterization of thyrocalcitonin. (Endocrinology 81: 306, 1967)

Calcitonin Activity of Particulate Fractions of the Ovine Thyroid Gland

“…These characteristics of the -175-unpurified hypocalcemic principle in the microsomal fraction are in accordance with the findings of Tashjian and Warnock (1967) in their studies with crude extracts of porcine CT.…”

“…Baghdiantz et al (1964), Hirsch et al (1964), and Tashjian and Warnock (1967) also demonstrated the peptide nature and the purconcomitant loss of biological activity of their relatively impure -CT preparations. These hypocalcemic preparations were readily in-.activated by incubating them with a variety of proteolytic enzymes such as trypsin and pepsin.…”

“…Serum calcium levels of rats receiving this preparation decreased approximately 29%, indicating no loss in hypocalcemic activity and the extreme thermostability of the microsomal preparation. Tashjian and Warnock (1967) had also shown that the timecourse of inactivation of -their crude porcine CT preparation by numerous proteolytic enzymes was extremely rapid. In order to demonstrate a similar effect with the hypocalcemic microsomal preparation, it was hydrolyzed for one hour at 37° C with pepsin at pH 2.7.…”

Calcitonin-Like Activity in the Circulation of Osteopetrotic Grey-Lethal Mice

“…It was shown that the hypocalcaemic activity was relatively stable to heat at neutral and acid pH. This has been reported to be a property of crude porcine calcitonin preparations (Tashjian & Warnock, 1967;Aldred & Schlueter, 1969). Pure preparations of porcine calcitonin are completely inactivated in vitro after incubation at 37°C in 0-1 M-tris (pH 7-5) for 4 h. Like the crude preparation of the hormone, the grey-lethal mouse plasma may contain some material which protects the calcium-lowering factor from inactivation.…”