PURIFICATION OF THE BRAIN HORMONE OF THE SILKWORM BOMBYX MORI HIRONORI ISHIZAKI AND MAMORI ICHIKAWA Department of Developmental Biology, Zoological Institute, College of Science, Kyoto University, Kyoto, Japan The endocrine function of the insect brain was first suggested by Kopec as early as in 1922 in the gypsy moth, Lymantria dispar. Years later, Wigglesworth (1940) clearly demonstrated that the molting of the bug, Rhodnius prolixus, was initiated by a hormonal factor originating from the dorsal region of the protocere-brum containing the neurosecretory cells. Since then, numerous studies have clearly defined the function of the insect brain hormone (BH). Thus BH stim-ulates the prothoracic glands to secrete the prothoracic gland hormone, ecdysone (e.g., Williams, 1947, 1952; Wigglesworth, 1952) which then is thought to act directly on the cells of the various tissues to provoke the growth and metamorphosis of the insect as a whole. BH thus occupies a central position in the endocrine net-work which controls the post -embryonic development of insects. The chemical study of BH has been reported from three laboratories with contradictory results. An extract possessing the BH activity was first prepared by Kobayashi and Kirimura (1958) from brains of the silkworm, Bombyx tnori. Later Kobayashi and his associates (Kobayashi et a!., 1962a, 1962b ; Kirimura et al., 1962; Saito et a!., 1963) obtained the active substance in a crystalline form and identified it as cholesterol. Gersch and his associates (Gersch et al., 1960) obtained the crystalline neurohormones from the entire central nervous tissue of the cockroach, Periplancta aniericana; the BH activity was detected in one of them, neurohormone D x (Gersch, 1961, 1962). In 1960 we obtained a potent watery extract from Bomby.v brains (Ichikawa and Ishizaki, 1960) and, later, con-cluded that this active principle was a protein (Ichikawa and Ishizaki, 1963). The present paper deals with the results of the further purification of BH. About 8000-fold purification, on the basis of protein measurement, was achieved and the hormone was proved to consist of chromatographically highly heterogeneous mole-cules, the molecular weight of the major components varying from 9000 to 31,000. MATERIALS AND METHODS Preparation of assay pupae Debrained pupae of the Eri-silkworm, Samia cynthia ricini, were used for the bioassay of BH. This species is non-diapausing but the arrest of development is brought about when the brain is surgically removed from pupae within 1 day after the pupal molt. BH was distinguished from the prothoracic gland hormone (ecdysone) by tests on isolated abdomens of S. cynthia ricini. The latter were prepared by cutting 355
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