Isolation and determination of the amino acid sequence of chicken calcitonin I from chicken ultimobranchial glands

Abstract

A radioimmunoassay for chicken calcitonin in chicken ultimobranchial glands was established utilizing a rabbit antiserum against eel calcitonin. This assay method, which is about 100 times as sensitive as the usual bioassay for hypocalcemic activity, was used for monitoring chicken calcitonin during its purification. The immunoreactivity in chicken ultimobranchial extract was separated by SP-Sephadex C-25 chromatography into two fractions. Chicken calcitonin I, which was occurred in the major immunoreactive fraction, was further purified to homogeneity as shown by reverse phase HPLC. In the end, 39 nmol of chicken calcitonin I was obtained from 3,384 chickens following a 12,000-fold purification. The complete amino acid sequence of purified chicken calcitonin I was determined to be H-Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Ly s-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2 and confirmed by synthesis. The specific biological activity of chicken calcitonin I (4,500 MRCU/mg) was identical to that of eel calcitonin, which has the highest specific biological activity among the calcitonins so far isolated. Chicken calcitonin I resembled the calcitonins from the ultimobranchial glands both of salmon and eel in sequence, biological activity, and immunological property.