Purification and some properties of prothoracicotropic hormone in the silkworm, Bombyx mori

Prothoracicotropic hormone was purified from the heads of Bombyx mori so that the nature of the hormone could be elucidated. The hormone extracted from acetone powder with 2% NaCl at pH 6.8 was partially purified by heat treatment, followed successively by ammonium sulphate precipitation, acetone fractionation and picric acid precipitation. Further purification was carried out by successive column chromatography on DEAE-cellulose, CM-Sephadex C-50, hydroxylapatite and Bio-Gel P-60. Finally, the hormone was isolated by preparative polyacrylamide gel disc-electrophoresis. These purification steps effected a 1700-fold increase in specific activity with a 4% yield of hormone preparation. From 1.5 × 106 heads, 5.3 mg of the hormone with a specific activity of 100,000 units per mg was isolated. The hormone labelled with 125I appeared as a single band with an apparent molecular weight of 18,000 on SDS-polyacrylamide gel electrophoresis. No loss of hormonal activity occurred in 6 M urea and 6 M guanidine hydrochloride solution. The isolated hormone was capable of inducing adult development of a debrained, dormant Bombyx pupa at a dose of 10ng, and was evidently different from the prothoracicotropic hormone (mol. wt 4400) obtained from the adult heads of Bombyx mori by Nagasawa et al. (1979) [Devl. Growth Differ.21, 29–38], which induced adult development of a brainless Samia pupa alone.