behavior in lepidopteran insects.1} EH was first isolated frompharate adult heads of the silkworm, Bombyx mori, and the sequence of the 13 amino-terminal.
Agric.
Biol
Chem.,
51 (8),
2307-2308,
2307
1987
steps of reverse phase HPLCusing Hi-Pore RP-304 (Bio Rad) with a gradient of aceto-
Short Communication AminoAcid Sequence of Eclosion Hormoneof the Silkworm, Bombyx mori
Takaharu Kono, Hiromichi Nagasawa, Akira Isogai, Hajime Fugo* and Akinori Suzuki Departmentof Agricultural Chemistry, Faculty of Agriculture, The Bunkyo-ku, UniversityTokyo of Tokyo, 113, Japan *Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183, Japan Received May 14, 1987
nitrile in 0.1% trifluoroacetic exchange chromatography
acid, anionusing TSKgel
DEAE-5PW(Toyo Soda) with a gradient of NaCl in 0.02m Tris-HCl buffer (pH 7.8) containing 10% acetonitrile, chromatography using
and reverse phase VP-318 (Senshu
Kagaku) or Hi-Pore RP-304 with a gradient of acetonitrile in 0. 1%heptafluorobutyric acid, two active fractions tained in the yield
"a" and "b" were obof 26.9jug and 10.3^g,
respectively. The specific activities of these active fractions were about 0.1 ~0.3ng/unit, and this value was equivalent to that of EH already isolated,3} suggesting that they were almost homogeneous. Amino-terminal amino acid sequences of the final preparations were analyzed using
0.8fig each by an Applied Biosystems Eclosion hormone (EH) is an insect neuro- about gas-phase sequencer (model 470A).
secretory behavior
hormone which triggers in lepidopteran insects.1}
eclosion EH was
Interestingly, it was found that both preparafirst isolated from pharate adult heads of the tions from fractions "a" and "b" contained silkworm, Bombyx mori, and the sequence of twotypes of amino-terminalsequencesof the the 13 amino-terminal amino acid residues was found to be H-Ser-Pro-Ala-Ile-Ala-SerSer-Tyr-Asp-Ala-Met-Glu-Ile-.2) Two forms
of EHs have been isolated from Bombyx adult heads, and another sequence two res-
idues shorter, starting from the alanine residue
at the amino-terminus, was found,3) indicating that the amino-terminal amino acid se-
quence of EH is heterogeneous. We purified EH from 770,000 Bombyx pharate adult heads (fresh weight 12kg) and analyzed its structure.
EH was purified by 18 steps, the first 14 like
those used in the purification from adult heads.3) After the 14th step, 118mg of the
25 or 23 amino acid residues shown in Fig. 1(1), which revealed 2-amino acid length heterogeneity similarly to EHs from adult heads.3) The EHs starting from alanine and serine residues isolated from fraction "a" were named EH-I and EH-II, respectively,
which seem to correspond to EH-I and EHII from adult heads. Similarly, the EHs with alanine and serine residues at the aminoterminus isolated from fraction "b" were
named EH-III and EH-IV, respectively.
For enzymatic digestion of EH, 500pmol of EH-III was reduced in Tris-HCl buffer
(pH 8.5) containing 600 nmol of dithiothreitol and 6m urea for 50min, and alkylated with 1200nmol of sodium iodoacetate for 20 min at
partially purified material with a specific activity of 170ng/unit2) was obtained. This ma- roomtemperature. Lysyl endopeptidase soterial was subjected to a Develosil C8 prepara- lution (l /xg/350/d 0.02m Tris-HCl (pH 9.7)) tive reverse phase HPLC column (Senshu was added to the reaction mixture, and the Kagaku) with a gradient of acetonitrile in resulting solution was maintained for 16 hr at 0.1% trifluoroacetic acid. The activity was 31°C. The digested peptides were separated by separated principally into two fractions "a" and "b." Each fraction was purified independentlv, and after three more purification
reverse
phase
HPLC using
VP-318
with
gradient of acetonitrile in 0. 1 %trifluoroacetic acid. Three major peaks consisting
a
of 17~20
2308
T. Kono et al. 1
5
H-Ser-Pro-Ala-ne-Al (1) (2) (3)
(h-^
(4)
10
15
a-Ser-Ser-Tyr-Asp-Ala-Met-Gl
u-I le-Cys-I
20
le-Gl u-Asn-Cys-Al
a-Gl n-
-7-)-r -rr -7T -7- -7 -^ --^ -P' -?> -^ -zr ^ -^- -^ ^ -^ h-,-^-^_,_,._,_^_,_^_,_^ ^ _^ _^^ |-^-^-^-^--,-^_,-^_^-_,_^_^ H3,1^ h^ -^
-^
-^-^
21
-^
-^
_^
25
_^
-_,
_^ _^ __^ L^ __,
30
Cys-Lys-Lys-Met-Phe-Gly-Pro-Trp-Phe-Gl (3)
-^
u-Gly-Ser-Leu-Cys-Al
41
45
a-Gl u-Ser-Cys-I
l e-Lys-
(2)
(4)
^-^
50
e-Pro-Gl
u-Cys-Gl
Hp- -^ -^ -^
I^
40
--^-^^^^^~~"~""^"^-^""~"""h^
Al a-Arg-Gly-Lys-Asp-n
I^
35
55
u-Ser-Phe-Al
a-Ser-I
^ _, -_,
60
l e-Ser-Pro-Phe-Leu-Asn-
_, _^ _,
^ -^ -^
(^^^^^^I^IJ-^-^'l^I^I^I^I^I^I^I^I^I^ 61 Lys-
(1) (2) (3) (4)
-^ -^
Fig. 1. Probable Arrow (-^) means Lysyl endopeptidase of EH-III. Dotted
61 Amino Acid Sequence of BombyxEclosion Hormone. the residues established by (1) Amino-terminal amino acid sequences of EH-I~IV. (2) digestion of EH-L (3) V8 protease digestion of EH-II. (4) Lysyl.endopeptidase digestion line (-) means that ho significant PTH-aminoacids were detected in the cycle.
amino acid residues each, ending at a lysine residue, were detected. Aminoacid sequences of
these
peaks
are
shown
in Fig.
1(4).
known. The sum of the molecular weight of
the 61 amino acid residues described above is 6,648.3. The calculated
molecular weight cor-
Furthermore, after re-chromatography of the flow-through fraction using another reverse phase HPLC column, NP-118 (Senshu
responds to about 80% of the 8,400 previously estimated from gel filtration.2) However, con-
Kagaku), the tetrapeptide shown in Fig. 1(4) was found, and this fragment was also supposed to originate from EH.
and the inaccuracy in estimation of molecular
To get the information that could connect the 4 sequences described above, EH-I and
EH-II were digested with lysyl endopeptidase and V8 protease after reductive respectively. Detected fragments quenced similarly (Fig. 1(2, 3)).
alkylation, were se-
EHs shown in Fig.
1(1),
weight
the sequencing
two of the
V8-protease-digested fragment corresponding to 37~48 shown in Fig. 1(3), affording the most probable 61(59) amino acid sequence of EH.
The exact molecular weight of EHis still not
we think
above
we have
found the full or nearly full sequence of EH. In surveying
the amino acid sequence homology
with knownpeptides and proteins, we couldn't find any peptide significantly homologous with EH.
Acknowledgment. This work was partly supported by for
Scientific
Research
(No.
61560135)
from the Ministry of Education, Science and Culture of Japan.
three major sequences detected from EH-III were thought to be connected with the supple-
ment of one lysine residue between the two fragments. The information about the order of the other two fragments was obtained from the
data described
by gel filtration,
a Grant-in-Aid
Considering the amino-terminal sequence of
natural
sidering
REFERENCES 1)
J. W. Truman, "Comprehensive
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Physiology,
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H. Fugo, and A. Suzuki, (1985).
Biochem., 15, 573
2)
H. Nagasawa,
T. Kamito,
S. Takahashi,
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A. Isogai,
3) H. Nagasawa, T. Mikogami, T. Kono, H. Fugo, and A. Suzuki,
Agric.
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