Eiji Ohnishi

Isolation and some characterization of the prothoracicotropic hormone from Bombyx mori.

The prothoracicotropic hormone (PTTH) was isolated from adult heads of Bombyx mori. Fifty micrograms of pure PTTH was obtained from 648,000 heads through a 15-step purification procedure with a 2 X 10(6)-fold purification and an 8% recovery. Chemical analyses of this PTTH have shown that it is a single-chain peptide consisting of 40-43 amino acid residues (MW, 4330-4740), the N-terminus of which is glycine. As little as 0.1 ng of PTTH elicited adult development in a debrained pupa of Samia cynthia ricini. Five picograms of PTTH directly stimulated the prothoracic glands in vitro so as to enhance ecdysone release. The hemolymph ecdysteroids of brainless Samia pupae that were developed by PTTH injection increased with essentially the same pattern as in developing normal pupae.

Changes in ecdysone titre during pupal-adult development in the silkworm, Bombyx mori

Abstract Changes in the ecdysone titre of the silkworm, Bombyx mori , during pupal-adult development were estimated. The average value of the maximum titre, which was observed on the second day after pupal ecdysis, was about 0·8 μg equivalent of β-ecdysone/g of live weight in both sexes. There is a distinct sexual dimorphism in the pattern of the ecdysone titre. The male exhibited a single sharp peak on the second day whereas the female showed the second peak on the fifth day. When the female was ovariectomized, the ‘female type’ ecdysone pattern was converted to the ‘male type’. In the female pharate adult 7 days after pupal ecdysis, ecdysone activity accumulated in the ovaries. The relationship between the ecdysone titre and adult differentiation, especially during ovarian development, is discussed.

Activation of prephenoloxidase: II. Activation by α-chymotrypsin

Abstract Activation by α-chymotrypsin of the prephenoloxidase, purified from the silkworm, Bombyx mori , was studied. The pre-enzyme was activated by the proteinase linearly with time without any lag period. The reaction velocity is proportional to the concentrations of α-chymotrypsin and of the pre-enzyme. The activation was suppressed by diisopropylfluorophosphate (DFP) and l -l-tosylamino-2-phenylethyl-chloro-methyl ketone (TPCK) and also by high concentrations of NaCl. The activated product seems to have a distinct molecular size as judged by gel filtration on Sephadex G-150, and showed a substrate specificity different from that of the activated enzyme produced by the action of the cuticular activator. Semialkaline proteinase of Aspergillus melleus showed an activating capacity nearly comparable to α-chymotrypsin. Neutral proteinase of Bacillus sublilis had slight activity, whereas, trypsin, leucine aminopeptidase, carboxypeptidase A, and alkaline proteinase of B. sublilis were inactive.

CONJUGATED ECDYSONE IN THE EGGS OF THE SILKWORM, BOMBYX MORI

A high activity of molting hormone (ecdysone) was detected in the extract of unfertilized, diapausing and developing silkworm eggs. The active substance in the diapause eggs behaved as a highly polar molecule at partitioning between n‐butanol and water, and also in TLC. The majority of the activity seems to be due to a conjugated ecdysone, since the polar active substance was changed to a less polar substance upon treatment with “snail enzymes”.

Accumulation of 2-deoxy-ecdysteroids in ovaries of the silkworm, Bombyx mori

Abstract 2-Deoxy-20-hydroxyecdysone was identified in the ovaries of pharate adults of the silkworm, Bombyx mori, by mass spectrometry and high performance liquid chromatography. Together with 2-deoxyecdysone, 2,22-bisdeoxy-20-hydroxy-ecdysone and ecdysone, this ecdysteroid constitutes one of the four major moulting hormones of Bombyx mori ovaries. Accumulation in ovary of these ecdysteroids suggests a forked pathway of biosynthesis, one leading to ecdysone and the other to 20-hydroxyecdysone.

Identification of estradiol in the ovaries of the silkworm, Bombyx mori

Estradiol was extracted and partially purified from the ovaries of the silkworm, Bombyx mori. Identification of estradiol was done by use of radioimmunoassay (RIA) and by gas chromatography-mass spectrometry (GC-MS) after derivatization into the ethyldimethylsilyl derivative. Concentration of estradiol in the ovaries was estimated to be 176 pg/g (RIA) and 63 pg/g (GC-MS).

Activation of prephenoloxidase 111. Release of a peptide from prephenoloxidase by the activating enzyme

Abstract Activation reaction of prephenoloxidase (pre-enzyme) was analyzed using a system of homogeneous pre-enzyme and highly purified prephenoloxidase-activating enzyme (PPAE) from the silkworm Bombyx mori . When pre-enzyme was activated by PPAE, release of a peptide was demonstrated. Results of polyacrylamide gel electrophoresis revealed that only a single peptide is liberated from pre-enzyme. Several lines of evidence indicated that the released peptide is inhibitory on PPAE.

BIOSYNTHESIS OF ECDYSONE IN THE ISOLATED ABDOMEN OF THE SILKWORM, BOMBYX MORI*

During pupal‐adult development of the silkworm, Bombyx mori, the ecdysone titer changes, exhibiting two maxima in the females: one on the second day of pupal development and the other just before adult emergence. During the second maximum, ecdysone accumulates in the ovaries. It also accumulates in isolated abdomens, which were prepared just after pupal ecdysis and induced to initiate adult development by injection with β‐ecdysone. Several lines of evidence suggest that ecdysone is synthesized in the ovary itself.

Isolation and identification of major ecdysteroid conjugates from the ovaries of Bombyx mori

Abstract Six major ecdysteroid conjugates have been isolated from mature ovaries of Bombyx mori by a procedure involving column chromatography on Sephadex G15, silicic acid and Sephadex LH-20, and high-performance liquid chromatography using a reverse-phase column. By analyses including ultraviolet absorption, enzymatic hydrolysis, negative ion fast-atom bombardment mass spectrometry and proton and 31 P nuclear magnetic resonance spectrometry, these conjugates were identified as the following: ecdysone-22-phosphate, 20-hydroxyecdysone-22-phosphate, 2-deoxyecdysone-22-phosphate, 2-deoxy-20-hydroxyecdysone-22-phosphate, 2,22-dideoxy-20-hydroxyecdysone-3-phosphate and bombycosterol-3-phosphate.

Occurrence of α-ecdysone in the developing embryos of the silkworm, Bombyx mori

Abstract From an acetone-ethanol extract of the developing embryos of the silkworm, Bombyx mori, the presence of α-ecdysone, but not of β-ecdysone, was shown by high pressure liquid chromatography and bioassay. The amount of α-ecdysone was estimated to be 0.74 μg per gram of eggs. The absence of a hydroxylating system at C-20 in the embryos is suggested.