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.

Sequence and expression of the chicken calcitonin gene

“…The nucleotide sequence of exon 4, which codes for calcitonin in this gene, is identical to the cDNA sequence of chicken calcitonin, and thus fully confirms the amino acid sequence deduced from the mRNA sequence. The deduced amino acid sequence of chicken calcitonin is also identical to the sequence of chicken calcitonin recently reported by Homma et al [19]. We observed a single difference between the genomic and mRNA sequences, i.e.…”

“…This difference could be due to polymorphism of this gene; Southern blot analysis of the chicken DNA extracted from several different animals of the same strain revealed that some of them had calcitonin/CGRP genes which lacked the EcoRI site. It is also highly probable that two calcitonin/CGRP genes exist in the chicken, inasmuch as a second calcitonin molecule was reported in chicken [19,20] and a second CGRP molecule exists in man and rat [21,221. Our hybridization data show that the specific mRNA for calcitonin is expressed predominantly in the ultimobranchial bodies and CGRP messenger principally in the nervous system. However, the calcitonin-specific probe did not detect RNA species longer than 1.5 kb.…”

Voltage‐gated sodium and potassium currents and their variation in calcitonin‐secreting cells of the chick.

“…7701R1) seemed to be high. This was presumably due to the close structural resemblance of eel calcitonin to that of the chick; only the 3rd and 4th N-terminal amino acids of the calcitonin amino acid sequence (total, thirty-two amino acids) are known to differ (Lasmoles, Jullienne, Desplan, Milhaud & Moukhtar, 1985; Homma, Watanabe, Hirose, Kanai, Kangawa & Matsuo, 1986). The sites showing these differences may not have been critical for the expression of antigenicity in the present preparation.…”

New Calcitonin Isolated from the Ray, Dasyatis akajei

“…1 ). As for function, non-mammalian calcitonins have a much greater hypocalcemic effect in the rat than do the mammalian calcitonins (Homma et al. 1986).…”

“…Although calcitonin-like immunoreactivity was also identified in the ultimobranchial glands of all classes of non-mammalian vertebrates (Van Noorden and Pearse, 1971;Tisserand-Jochem et al. 1977;Sasayama et at., 1984;Treilhou-Lahille et al, 1984), structures have been determined for only one species of bird and two teleost fishes (Niallrta/.. 1969;NodaandNarita, 1976;Homma et al. 1986).…”