Bombyx PTTH

Prothoracicotropic hormone (Bombyx PTTH) of the silkworm, Bombyx mori, was extracted from adult heads. Bombyx PTTH is suggested to be a glyco-peptide and consists of two essentially identical subunits. Amino acid sequencing and cDNA analysis revealed the whole amino acid sequence of the subunit, composed of 104-109 residues. Bombyx PTTH stimulated adult development in brainless Bombyx pupae at a dose of ca. 0.1 ng and also enhanced the release of ecdysone in vitro at a concentration of 10 M. Immunohistochemistry and in situ hybridization showed that Bombyx PTTH was transcribed and translated in two pairs of dorso-lateral neurosecretory cells in the brain. 

During purification of Bombyx PTTH, samples were bioassayed using the debrained pupae of racial hybrid J-122 x C-115 of Bombyx, and their PTTH activities were expressed in terms of Bombyx units as described previously (10). After many trials, one molecular species of Bombyx PTTH was isolated through 16 steps of purifications (11). Heads were homogenized successively with cold acetone (step 1) and 80% ethanol (step 2), and the residues were extracted with 2% NaCl (step 3), and the extract was heated in boiling water to remove the resulting precipitates (step 4). 

At step 15, PTTH activity was recovered in four consecutive fractions, each of which corresponded to a UV peak at 280 nm, and the most active fraction was further purified at step 16 to afford four active fractions with nearly equal specific activities (0.1 nq/Bombyx unit). finally, a fraction that gave a single amino-terminal sequence was obtained. These results indicated that Bombyx PTTH was highly heterogeneous. The amount of Bombyx PTTH isolated from the most prominent peak at step 16 was only 5.4 ug from 500,000 Bombyx heads. 

Amino Acid Sequence Analysis of Bombyx PTTH... 

Finding the glycosylation site suggests that Bombyx PTTH may be a glycoprotein as previously indicated (13). The heterogeneity of Bombyx PTTH may be partly due to the presence of a glycosidic side chain. 

The Bombyx PTTH was active at a dose of ca. 0.1 ng, when injected into brainless Bombyx pupae, but was completely inactive in debrained Sarnia pupae even at a dose of 10 ng (11). The Bombyx PTTH also enhanced the release of ecdysone by the prothoracic gland of Bombyx in vitro at a concentration of... 

Figure 2. Schematic representation of pre-pro-Bombyx PTTH cDNA. Coding regions are indicated by boxes numerals in parenteses are the number of amino acid residues in the respective components. Putative proteolytic cleavage sites are indicated by KRK, KR, and RKR. Triangles represent the polyadenylated tract.

We used the Bombyx adult heads as a source of PTTH (5) along with bombyxins (formerly 4K-PTTH, insulin-related peptides possessing PTTH activity in the brainless pupae of Sarnia cynthia ricini) (6), eclosion hormone (7), melanization and reddish coloration hormone (8) and pheromone biosynthesis activating neuropeptide (9). All these peptide hormones have been isolated and characterized. 

Further, in situ hybridization using S-labeled complementary RNA probe showed that the Bombyx PITH gene was transcribed in the same cells, indicating that Bombyx PITH is synthesized in these cells (12). In Manduca sexta two pairs of brain neurosecretory cells in a similar location have been immunohistochemically identified as the PTTH-producing cells (14). 

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