|Budget Amount *help
¥6,900,000 (Direct Cost : ¥6,900,000)
Fiscal Year 1992 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1991 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1990 : ¥4,900,000 (Direct Cost : ¥4,900,000)
From two species of amphibians (anura: Rana catesbeiana, adults (N=700), tadpoles (N=4,000); urodela: Cynops pyrrhogaster, adults (N=6,000)), ultimobranchial glands whichi are the source of calcitonin were dissected out. The number of animals are necessary to be purified for calcitonin was determined by rat-bioassay. In the two species, the homogenate of the ultimobranchial gland was centrifuged, and its precipitate was suspended in 0.1M acetidc acid. After centrifugation, the supernatant was then subjected to reversed-phase HPLC with linear gradient elution from 20 to 80 % acetonitrile in 0.1 % trifluoro acetic acid. Then, a part of each fraction was examined for the presence of immunoreactive calcitonin by means of immunobloting. Then, a protion of the sample was subjected into SDS-PAGE. The elution in HPLC obtained two immunoreactive fractons, at 33 % and 44 % position in acetonitrile concentration. The former fraction was further subjected into HPLC. After performing it twice, a si
ngle peak was obtained, which was probably calcitonin as checked by means of the Western blotting after SDS-PAGE. The molecular weight of the substance corresponded to about 3,500. The fraction of 44 % obtained an immunoreactive portion, but not a sharp peak. Its molecular weight corresponded to about 10,000-13,000 after SDS-PAGE. It was probably the prohormone of calcitonin. The single peak of 33 % position was sequenced in those two species However, the quantity of the purified hormone was not enough for its amino acid sequence to be determined. It was suggested that the amphibian calctionins purified in the present study were smaller than the considered amount. There- fore, the amphibian calcitonins may have a higher potency than the salmon calcitonin, having the largest potency in calcitonins. On the other hand, in the newt, in the process purification, two peaks which contained newt nuclear histones were obtained. The newt histone was found to be more homologous with the African clawed toad, an anuran amphibian, H2B.2 hostone, than with the other known H2B histones.