122 x C-l 15 of B. mori and their activities were expressed in terms of Bombyxunits as described.10) ...... M. K. Thomas,. N. Agui and. A. H.. Baumhover, Science ...
Agric.
Biol.
Chem.,
51 (4),
1067-1076,
1987
1067
Isolation and Partial Characterization of a Prothoracicotropic Hormone of the Silkworm, Bombyxmori
Hiroshi Kataoka, Hiromichi Nagasawa, Akira Saburo Tamura, Akira Mizoguchi,* Yuko Fujiwara,* Chu Suzuki,* Hironori Ishizaki* and Akinori Suzuki
Isogai,
Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan * Biological Institute, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464, Japan Received October 8, 1986 Onemolecular species ofprothoracicotropic hormonewith a molecular weightof about 22,000 (22K-PTTH) of the silkworm, Bombyxmori, was isolated from 5 x 105 adult heads. The purification procedure consisted of 16 steps including defatting, salt-extraction, fractional precipitations, conventional columnchromatographies, and high performanceliquid chromatographies. An
approximately 5 x 106-fold purification with
a recovery
of 3.3%.
was attained to yield 5.4 fig (0.25 nmol) of the pure hormone
Injection
of 0.1Ing
of the purified
22K-PTTH
could
elicit
adult
development in a brainless Bombyx pupa. 22K-PTTH is a basic protein (pi 7.7~8.7) containing disulfide bond(s). The amino-terminal amino acid sequence of 22K-PTTHwas determined to be Gly-Asn-Ile-Gln-Val-Glu-Asn-Gln-Ile-Pro-Asp-Pro-.
Prothoracicotropic
hormone (PTTH),
the
insect brain hormone that stimulates the synthesis and release of ecdysone in prothoracic glands,1>2) has been purified from brains or whole heads of several insect species.3~9) The adult heads of the silkworm, Bombyx mori, contain two distinct species-specific PTTHs: 22K-PTTH (or PTTH-B) active on brainless
pupae of Bombyxbut not on those of Samia cynthia ricini, and 4K-PTTH (or PTTH-S), on the contrary, active on Samia but not on Bombyx}0) Therefore, 22K-PTTH is now considered to be a genuine PTTH in Bombyx but the intrinsic function of 4K-PTTHin Bombyx remains unknown. 4K-PTTHhas been characterized as a heterodimeric peptide consisting of A- and B-chains associated by disulfide bonds, and one of the various molecular forms of 4KPTTHs has been fully
sequenced.n)
On the
other hand, 22K-PTTH, possibly the same molecule
which
has
Kobayashi and his colleagues3'
been
studied
by
12) for nearly 30
years, still
remains to be isolated
in a pure
form so that its chemical nature can only be assessed using the partially purified preparation. We established that 4K-PTTH and 22KPTTH were completely different molecular species on the basis of the results obtained
from acetone fractional precipitation,
gel fil-
tration, and ion-exchange chromatography,13) and also attempted to purify 22KPTTH using a Bombyx pupal assay.10) We
report here the isolation characterization amino-terminal
of 22K-PTTHand its
by amino acid analysis sequence analysis.
and
MATERIALS AND METHODS
/. Experimental procedure and chemicals. Extraction, fractional precipitation, and conventional liquid chromatography were done in a cold room (4°C) or in an ice-water bath. High performance liquid chromatography (HPLC) was done using
a TRI
ROTAR-III
HPLC system
(JASCO) at 40°C. The amount of protein in each fraction was measured by the absorption at 280nmor 220nmwith
1068
H. Kataoka et al.
bovine serum albumin (BSA) as the standard. Glass tubes used for fractionation at steps 15 and 16 were specially cleaned to removeany contaminants: they were washed with glass-distilled water, ultrasonicated in 50% nitric acid at room temperature
for 30min,
then rinsed
repeatedly
with water for HPLCuntil the pHof the rinsing became neutral. All chemicals
used were of the highest
mercially available. Water was distilled
grade
com-
after permeation
and deionization. Trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA) were the reagents for amino acid sequencing (Wako). Water and acetonitrile
used for HPLCwere purchased from Nakarai and CicaMerck, respectively. 2. Purification of22K-PTTH Source of 22K-PTTH. The source material, male adult heads of the silkworm, Bombyxmori, was the same as used for purification of 4K-PTTH.14)
was then repeated for the other half of the acetone precipitates. Step 8. Anion-exchange on DEAE-Sepharose. The active zones from the two runs of gel filtration were combined to give a final volume of 800ml and applied to a
column (55 x 80mm) of DEAE-Sepharose CL-6B equilibrated
with
0.2m
sodium
acetate
buffer
(pH
7.0).
The
column was washed with 400ml of the same buffer. Step 9. Cation-exchange on CM-Sepharose. The unadsorbed fractions from the DEAE-Sepharose column were pooled, adjusted to pH 5.2 with acetic acid, diluted four times with distilled water, and applied to a column (31 x 400mm) of CM-Sepharose CL-6B which had been equilibrated with 0.05 m sodium acetate buffer (pH 5.2). The column was eluted sequentially with 500ml of the same buffer, 500ml of 0.1 m NaCl in the same buffer, a linear gradient of 0.1 ~0.5 m NaCl in the same buffer (500 ml each), and finally with 500ml of0.5 m NaCl in the same buffer. The flow rate was 60ml/hr and 20-ml fractions
Bioassay. The samples after each purification step were were collected. Step 10. Hydrophobic chromatography on Octylbioassayed using brainless pupae of a racial hybrid J122 x C-l 15 of B. mori and their activities were expressed Sepharose. The active fractions from CM-Sepharose chromatography were pooled (400ml), and 123g of solid in terms of Bombyxunits as described.10) Test materials were routinely lyophilized once, then dissolved in 0.1 m ammoniumacetate was added with gentle stirring to give a solution of about 4m. This solution was loaded on a phosphate buffer (pH 7.3) containing 0.1% BSA. After CL-4B at a flow reversed phase HPLC, samples for bioassay were added to column (35 x 330 mm) ofOctyl-Sepharose the above buffer before lyophilization. BSAminimizes rate of 20ml/hr, which had been equilibrated with 4m losses tainers acidic acidic
due to non-specific adsorption to the glass conand the phosphate buffer served to neutralize the effluent because 22K-PTTH is somewhat unstable in solution.
ammonium acetate. Column eluents were (A) 4m ammonium acetate, (B) 0.2m ammonium acetate, and (C) 40% acetonitrile in 0.2 m ammoniumacetate. Elution was made successively with 250 ml (A), a linear gradient of (A) to (B) (300ml each), 200ml (B), a linear gradient of(B) to Steps 1~5. 22K-PTTHwas extracted from a total of (C) (300ml each), and 300ml (C) at a flow rate of50ml/hr, 5 x 105 heads of male adult Bombyx (fresh weight, 3.75 kg) and 15 ml fractions were collected. Step ll. Gel-filtration on Sephadex G-75. The active in four batches and purified partially up to step 5 by the same procedure as used for purification of 4K-PTTH15): fractions from Octyl-Sepharose chromatography were successive washing with acetone (step 1) and 80%aqueous combined and concentrated to about 20ml by ultra filethanol (step 2), extraction of 22K-PTTH with 2% NaCl tration using an AmiconYM-10membranewith a mo(step 3), heat-treatment (step 4), and precipitation with lecular weight cutoff of about 1 x 104. The retentate was lyophilized, redissolved in 2ml of 0.5 m Tris-HCl buffer 80% saturation of ammonium sulfate (step 5).
Step 6. Acetone precipitation. The pellets after precipitation with ammoniumsulfate were dissolved in 800 ml of distilled water, to which 430ml of cold acetone was added with gentle stirring to give a 35%acetone solution. The precipitates formed were removed by centrifugation at 3200g at 4°C for 20min. Then 540ml of cold acetone was added to the supernatant, resulting in a 55% acetone solution. The precipitates with PTTHactivity were obtained by centrifugation under the sameconditions. Step 7. Gel-filtration on Sephadex G-50. The 35~55% acetone precipitates derived from 2.5 x 105 heads were dissolved in 200ml of 0.5 m Tris-HCl buffer (pH 8.5) and applied to a column (5.5 x 40cm) of Sephadex G-50 (fine) previously equilibrated with 0.2 m sodium acetate buffer (pH
7.0)
containing
2% «-butanol.
The materials
were
eluted with the same acetate buffer at a flow rate of200 ml/ hr and 20 ml fractions were collected. The same procedure
(pH
8.5),
and applied
to a column
(25x610mm)
of
Sephadex G-75 (super fine) equilibrated with 0.01 m phosphate buffer (pH 6.5) containing 0.2m NaCl and 2% n-
butanol. The column was eluted with the same phosphate buffer at a flow rate of 10ml/hr and 4-ml fractions were collected.
Step 12. Reversedphase HPLCon Develosil 5C8. The lyophilized
filtration applied
active
material
after
Sephadex
G-75 gel-
was dissolved in 10ml of water for HPLC, and
to a 20x300
mm column of Develosil
5C8
(Nomura Kagaku) at a flow rate of 1 ml/min. Then the materials were eluted with a gradient of 20~50%acetonitrile in 0.08% TFAin 120min at 40°C at a flow rate of 2.5 ml/min. Fractions of 5 ml were collected. Step 13. Reversed phase HPLC on Hi-Pore RP-304. The active fractions from the previous step were pooled and lyophilized after addition of 50jA of 1 m NaCl. This
Prothoracicotropic Hormoneof the Silkworm
1069
was redissolved in 2ml of HPLCwater and subjected to a amide in distilled water was added to the reaction mixture, 4.6x250mm column of Hi-Pore RP-304 (Bio-Rad) which was then kept in the dark under nitrogen at room
equilibrated
with 20% acetonitrile
in 0.08% TFA. The
column was eluted with a linear gradient of 20~40% acetonitrile in 0.08% TFA at a flow rate of 1 ml/min and 1ml fractions were collected. Step 14. Reversed phase HPLC on Hi-Pore RP-304. All fractions with PTTH activity from the previous step were pooled and lyophilized after the addition of 50/il of 1 m NaCl. It was then redissolved in 1 ml ofHPLCwater and loaded onto the same column as used at step 13, which had been equilibrated with 20% acetonitrile containing 0. 1% HFBA.The materials were then eluted with a 40-min linear gradient of20 ~40% acetonitrile in 0.1% HFBA at a flow rate of 1 ml/min and 1-ml fractions were collected. Step 15. Cation-exchange HPLC on TSKgel SP-5PW. The active fractions from the previous HPLC were pooled (4ml), and 160/il of 0.5m sodium acetate buffer (pH 5.2) was added to make a 0.02m buffer. This was applied to a 7.5x75mm column of TSKgel SP-5PW (Toyo Soda) previously equilibrated with 0.02 m sodium acetate buffer (pH
5.2)
containing
10%
acetonitrile.
A 50-min linear
gradient of 0.02~0.5m sodium acetate buffer (pH 5.2) in 10% acetonitrile was run through at a flow rate of
1 ml/min. Each UVpeak monitored by the absorbance at 280nmwas collected and bioassayed. Only the material from the second (tR 31.2~31.9 min) of the four peaks with activity (tR 30.5~34.3 min in Fig. 3a) was purified further.
Step 16. Reversed phase HPLC on Hi-Pore RP-304. The active material from the previous step was directly loaded onto the column of Hi-Pore RP-304 used at steps 13 and 14, equilibrated with 20% acetonitrile in 0.1% HFBA.The column was eluted with two successive gradients of 20-32% acetonitrile containing 0.1% HFBA in 5 min and 32 ~40% acetonitrile containing 0.1% HFBA in
temperature
for 20min.
3.
Chemical characterization
bio-
assayed without desalting. A control experiment without
dithiothreitol and iodoacetamide was also done. Isoelectric focusing. Flat-bed preparative isoelectric
focusing was done using the Multiphor system (LKB), Sephadex-IEF (Pharmacia), and Ampholine (pH 3.5 ~ 10, LKB). The partially
purified
preparation
of 22K-PTTH
(after step 9, 2.6 x 104 Bombyx units) was loaded in 3ml of 2%Ampholine solution near the anode. Constant power of 8Wwas applied for 15hr. Then the bed was divided
across the pH gradient into 30 equal parts, each of which was extracted with 2 ml of distilled water. After measuring the pH of each fraction, the gel was again extracted with 1 ml of0.2m NaCl. The two extracts of each fraction were combined,
lyophilized,
and bioassayed
directly
without
removing Ampholine.
Amino acid analysis. The purified 22K-PTTH (1 jug) was hydrolyzed in HC1 vapor using a PICO TAGWork Station (Waters) at 1 10°C for 20hr. The hydrolyzate was analyzed after amino acids16)
derivatization to phenylthiocarbamoyl on a TRI ROTAR-III HPLC system
(JASCO) with a Senshu Pak ODS-1151-N column (4.6 x 150 mm). Elution
acetonitrile
was done by a gradient
of 2~50%
in 10mMphosphate buffer (pH 5.5) at 47°C
with a flow rate of 1 ml/min. The effluent was monitored by measuring the absorbance at 254 nm. Amino acid sequence analysis. The purified 22K-PTTH (about 4fig) was sequenced by an automated gas-phase
sequencer (Applied Biosystems, model 470A) and a PTH amino acid analyzer (Applied Biosystems, model 120A). The phenylthiohydantoin (PTH) amino acids obtained after each cycle from the sequencer were introduced directly into the analyzer.
80min, at a flow rate of 1 ml/min.
Trypsin digestion.
The mixture was directly
RESULTS
of22K-PTTH
TPCK-trypsin (Sigma) was dissolved
in 0.1 m Tris-HCl buffer (pH 8.0) containing 0.01 m CaCl2 at a concentration of 2mg/ml. The enzyme solution (200jul) was added to the lyophilized preparation after purification step 10 (1 x 104 Bombyx units). The mixture was incubated at 28°C for 3 hr with vigorous shaking. The digestion was ended by heating it in a boiling water bath for 5min. A control experiment without the enzyme was also done. a-Chymotrypsin digestion. 22K-PTTH was digested by a-chymotrypsin (Sigma) in exactly the same way as with TPCK-trypsin except that the buffer did not contain CaCl2.
Reduction and alkylation. The partially purified preparation of 22K-PTTHafter step 10 (1 x 104 Bombyx units) was dissolved in 50^1 of 0.5m Tris-HCl buffer (pH 8.4) with 6 murea containing 2 m dithiothreitol, and incubated under nitrogen at 45°C for 1 hr. Then 50fd of4m iodoacet-
1.
Isolation
of22K-PTTH
The 16-step procedure of the extraction and purification of 22K-PTTHfrom Bombyxadult heads as well as the yields and activities after
each step are summarized in Table I. Steps 1 ~6. As reported previously,13)
22K-
PTTH was extracted successfully with 2% NaCl from Bombyx adult heads. The saline
extracts from 5 x 105 heads had an activity of 1.5x 106 Bombyx units, i.e., an adult head contains 3 Bombyx units. Since 22K-PTTH
was shown to be heat-stable and to precipitate in 80%saturation of ammoniumsulfate,13) the same purification procedure as for 4K-PTTH was used in the initial five steps. The previous report13)
also showed that acetone fractional
1070
H. Kataoka et al.
Table I. Summary of Purification P
urific ti n ste
of 22K-PTTH from 5 x 105 Bombyx Adult Heads
Weight
un caion sep
^^
Total activity
Bombyx heads 3.75 x 106 1. Washingwith acetone 2. Washing with 80% ethanol 3. Extraction with 2% NaCl 850,000 4. Heat treatment
5. 6. 7. 8. 9. 10. ll. 12. 13. 14. 15.
the highest
*
peak
peaks peak (HFBA) peaks
1,500
570,000
450 150
0.015
140
(100)*
-
1,500 1,500 1,500 1,500 1,300 1,000 1,000 900 750 500
0.20 0.043
(%)
-
-
0.0054
Yield
(ng/Bombyxunit) -
-
Precipitation with (NH4)2SO4 200,000 Precipitation with acetone 40,000 Sephadex G-50 30,000 DEAE-Sepharose 6,400 CM-Sepharose 1,000 Octyl-Sepharose 300 Sephadex G-75 100 Develosil 5C8 14 Hi-Pore RP-304 (TFA) 5.3 Hi-Pore RP-304 (HFBA) 1.4 TSKgel SP-5PW
total of 4 the second 16. Hi-Pore RP-304 total of 4
Specific activity
(Bombyxunitsx10~3)
-
130,000 26,000 20,000 4,300 770 300 100 16 7.1 2.8
100 100 100 100 87 67 67 60 50 33
0.44 0.28
30 10
0.ll
50
9.3
0. 1 1
3.3
Nominal.
after centrifugation. Thus, two precipitation separated 22K-PTTHfrom 4K- precipitates PTTH: 22K-PTTH precipitated in 40-57% steps of purification were neccessary as a preacetone
and 4K-PTTH in more than 57%
treatment
for
CM-Sepharose
chromatog-
acetone. In this experiment 22K-PTTHwas raphy. At step 7, gel filtration on Sephadex G50 was done only to remove ammoniumsulrecovered in the 35 - 55%acetone precipitates, fate in the acetone precipitates. As a column which weighed about 40g, and was active at a dose of 26fig. Almost no loss of activity was eluent we selected 0.2 m sodium acetate buffer (pH 7.0) so that the active fractions obtained observed during steps 3 -6. Steps7~11. Steps 7-ll consisted of con- from this chromatography could be used diventional liquid chromatographies. Prelimi- rectly in the next step. At step 8, the active nary experiments demonstrated the effectivefractions from gel filtration were applied to a ness of CM-Sepharose, but the acetone pre- DEAE-Sepharose column. As expected from cipitates could not be applied directly to our previous study,13) 22K-PTTH broke CM-Sepharose because the precipitates con- through the column and most acidic proteins, tained ammoniumsulfate, the amount differ- which would be insoluble in the initial buffer in were ading considerably depending on the run, caus- CM-Sepharose chromatography, ing poor reproductibility in the buffer concentration at which 22K-PTTH was eluted
sorbed to the DEAE-Sepharose. Most of the
colored
(dark
violet)
materials
were also ad-
from CM-Sepharose, and also because the sorbed to this ion-exchanger, which resulted in precipitates could not be dissolved completely decoloration of the unadsorbed fraction con-
in 0.05m sodium acetate buffer (pH 5.2), the
taining
initial
ments, the active fraction was used in CM-
buffer
in CM-Sepharose
chromatog-
22K-PTTH.
Following
these
treat-
raphy, and in that case PTTHactivity was Sepharose chromatography (step 9). 22Kusually found in both the supernatant and the PTTH was adsorbed to CM-Sepharose and
Hormoneof the Silkworm
1071
a
Prothoracicotropic
A
2-
h
si-
-''
+*?o
\/---"m\
oL^
0.8-
"°*6^
*J\\
.
å °"3
7^»H*^»i
b
^
^""lo
'4!
Jo*
f\
/
\ y
^^Y/p//////////M(//rf/7M/ i
A
50
C
§
1/
\
\
§
1 \
O
100
150
on^ *^"S
°
I I
Vo/
^^^.
æf/
0 I-//^^
1
V///////////////A
"
^*^^^s_
1
30
L_
50 Fraction
1
1
70 no.
Fig. 1. Purification of 22K-PTTH by Conventional Liquid Chromatographies. Cross-hatched bars indicate PTTHactivity, (a) Ion-exchange chromatography on CM-Sepharose CL-6B. 22K-PTTHwas eluted by a linear gradient of 0.1 -0.5m NaCl in 0.05m sodium acetate buffer (pH 5.2) illustrated by a dotted line, (b) Hydrophobic chromatography on Octyl-Sepharose CL-4B. Concentrations of ammoniumacetate and acetonitrile are represented by dotted and solid lines, respectively, (c) Gel-filtration on Sephadex G-75 (super fine). As an eluent 0.01 m phosphate buffer (pH 6.5) containing 0.2m NaCl and 2% nbutanol was used. Vo indicates the void volume of this column.
eluted as a single broad peak in a range of 0.25~0.4M NaCl (Fig. la).
materials and those eluted with more than 10% acetonitrile could be removed. the Then the active fraction was processed to After concentration and lyophilization, hydrophobic chromatography (step 10). The Octyl-Sepharose active fraction was applied to flow rate was strictly controlled, because pre- gel filtration on Sephadex G-75 (step ll). liminary experiments showed that the main- PTTH activity appeared in fractions 43~54 tenance of a sufficiently slow and steady flow (Fig. lc), which is consistent with the previous rate gave good resolution and reproducible molecular weight estimation of about 22,000 by gel filtration on Sephadex G-50. The active results. As shown in Fig. lb, PTTHactivity was eluted over a wide range (fractions materials thus obtained had a specific activity 80~ 130), but a large amount of unadsorbed of 100 ng/Bombyx unit. This 6-step procedure
1072
H. Kataoka et al.
^
A
I
]
'
UO.
'
b
80
yv^.
1-
°°
--""""405m
\
----""""
^
-40
\
-
. on
\
K
/uU
20
40
20 Retention
time
40 (min)
Fig. 2. Reversed phase HPLC on (a) Develosil 5C8 (step 12), and (b) and (c) Hi-Pore RP-304 (steps 13 and 14). Cross-hatched bars indicate PTTHactivity. Dotted lines represent the concentrations of acetonitrile TFA (steps 12 and 13) and in 0.1% HFBA (step 14).
by liquid chromatographies effected a 260-fold purification with a 67% recovery.
Steps 12~16. The final purification was made by five steps of HPLC. After many attempts to purify 22K-PTTH by reversed
phase HPLC,we selected columns of Develosil 5C8 and Hi-Pore RP-304. Because the amount of the Sephadex G-75 fraction was too large (100 mg), it was first run through a preparative column (20 x 300mm) of Develosil 5C8 (step
in 0.08%
TFA) of the UV peaks (Fig. 2b). At step 14, the 22K-PTTHfractions were applied to the column used in the previous step, but the solvent system was changed from TFA/
acetonitrile to HFBA/acetonitrile, which we considered more suitable for separating basic peptides or proteins. Basic proteins are generally eluted at a higher concentration of
acetonitrile in HFBAthan in TFA. Actually, 22K-PTTHwas eluted from 33 to 36% aceto12). 22K-PTTH was eluted from 33 to 37% nitrile in 0.1% HFBA, about 7% higher than acetonitrile in 0.08% TFA (Fig. 2a). This by TFA at step 13 (Fig. 2c), suggesting that
HPLCattained a 6-fold purification
and yield-
latter
in 0.08%
22K-PTTH is a basic protein. The pi value of 22K-PTTH was 7.7-8.7, as described later. applied to a column of Hi-Pore RP-304 (step 22K-PTTHfractions were then applied to a 13). PTTH activity was recovered from the column ofTSKgel SP-5PW (step 15). This ioned 14mg of active
part
(26-29%
material.
acetonitrile
This
was then
exchanger is a large-pored
(1000A)
synthetic
Prothoracicotropic
polymer and is suitable for separation of proteins because of high recovery. PTTHactivity was recovered from four peaks between 0.2 and 0.3m sodium acetate buffer with a total activity of 4.5 x 105 (Fig. 3a). The recovery at this
step was about 90%. Among the four
peaks, only the fraction corresponding to the second earliest eluted peak, which was sharpest in profile and had the highest specific activity (0.28 ng/Bombyx unit), was purified
further. At step 16, the final step, the column and
solvent system used at step 14 were used, but elution was done by a muchshallower gradient of acetonitrile (0.1%/min) to separate many crowded peaks with similar hydrophobicity as as possible'.
PTTH activity
appeared
again in four peaks with a total activity of 1.4 x 105 Bombyx units (Fig. 3b). The recovery was about 93%. Though all these four peaks had almost equal specific activity (0.10 ~0. 15 ng/Bombyx unit), only the highest
peak was big enough to characterize chemically. The amino-terminal sequence of a portion of the material from the highest peak was analyzed; there was a single amino acid sequence, as described later. Furthermore, this material showed a single peak by rechromatography under the same conditions, though
some small peaks probably derived from im-
purities in the HPLCsolvents were observed (Fig. 3c). These data indicated that the material was sufficiently pure. Therefore, the isolation of one molecular species of 22K-PTTH
a
finely
1073
Hormoneof the Silkworm
20
40
b 0.05
-
A
A
/\
å 40S
20
0.002
å
S
20
3. Purification
*
40
40
Retention
Fig.
*f'
time
(min)
of 22K-PTTH by HPLC.
Cross-hatched bars and area indicate PTTHactivity, 15). 22K-PTTHwas eluted by a gradient of0.02~0.5
(a) Cation-exchange HPLCon TSKgel SP-5PW(step
m sodium acetate buffer,
pH 5.2, shown as a dotted line,
in 10% acetonitrile. (b) Reversed phase HPLC of the second active peak (tR 31.2~31.9.min) from (a) on HiPore RP-304 (step 16). An extremely shallow gradient of acetonitrile (increasing at 0. 1 %/min) in 0. 1 % HFBA, represented by a dotted line, was done, (c) Rechromatography of a portion of the material ffbm the highest peak in (b) under the same conditions as in (b).
1074
H. Kataoka et ai
was considered to be accomplished. The final 5-step procedure by HPLC effected a 150-fold
by a gas-phase sequencer with on-lined HPLC for analysis of phenylthiohydantoin derivapurification and yielded 5.4/ig (0.25nmol tives of amino acids revealed that the aminobased on the molecular weight of 22,000). As terminal amino acid sequence of 22K-PTTHis
shown in Table I, the total recovery from step 3 to step 16was 3.3% and a 5 x 106-fold purifi-
Gly-Asn-Ile-Gln-Val-Glu-Asn-Gln-Ala-Ile-
tivity at a dose of0.ll ng.
80.5,
Pro-Asp-Pro- (The amounts of PTH-amino cation was attained. The purified 22K-PTTH acid recovered from each cycle "of Edman obtained from the highest peak showed ac- degradation were 102.5, 137.5, 129.5, 120.5,
2. Chemical characterization Incubation PTTH with
of 22K-PTTH
of the partially purified 22Ktrypsin or a-chymotrypsin de-
creased its activity to 1%or less than 1%of the
original, respectively, indicating the proteinous nature of 22K-PTTH. Reduction of 22K-PTTH with dithiothreitol followed by alkylation with iodoacetamide resulted in nearly complete loss of activity, suggesting that
22K-PTTH contains disulfide bond(s) essential for activity. The results from isoelectric focus-
ing of 22K-PTTH indicated that its pi is 7.7-8.7. This basic nature of 22K-PTTH is in
accordance with its behavior on ion-exchange chromatographies at steps 8, 9, and 15. The results of amino acid analysis of the purified 22K-PTTH is shown in Table II. It
has large proportions of aspartic acid or asparagine, glutamic acid or glutamine, and proline residues, and small proportions of methionine
and phenylalanine Edman degradation
residues. The automated of the purified 22K-PTTH
82.0,
52.6,
51.3,
80.8,
48.5,
34.0,
15.0,
and
28.0 pmol). The sole amino acid sequence
assured the purity of the final preparation. DISCUSSION
22K-PTTH was extracted from 5xlO5 Bombyx adult heads and purified to homogeneity by a 16-step procedure. 22K-PTTH
was separated from 4K-PTTHby fractional precipitation with acetone at step 6, as expected from the previous studies.13) Several conventional
liquid
chromatographies
purify 22K-PTTHconsiderably,
served to
but were not
efficient enough to isolate a pure hormone. This situation led us to examine the purifi-
cation by HPLCbecause of its high resolution and reproducibility. Quite recently, some packed columns of good quality for separation of proteins have become commercially available. Among them, Hi-Pore RP-304 and
TSKgel SP-5PWcolumns gave particularly
good results. After searching for the best HPLCconditions such as solvent systems and
elution methods, further 5-step HPLCpurifiTable II.
Amino Acid Composition
of 22K-PTTH
Amino acid
Percent*
Amino acid
Percent
Asx Glx
1 1.8 17.1
Tyr Val
Ser
7.0
Met
0.2
Gly
4.6
Cys2
0.5
His Thr Ala Arg
2.0 7.2 4.4 6.8
He Leu Phe Trp
6.5 7. 5 0.8 -**
Pro.
9.0
Lys
5.8
3.9 5.8
Hydrolysis was done in HC1 vapor at 110°C for 20hr. * Values were obtained from a single analysis and
represented as percentages. N* Decomposed during acid hydrolysis.
cation
procedure
could
be established.
The
overall 5 x 106-fold purification yielded only 5.4fig (0.25 nmol) of one molecular species of 22K-PTTH, the recovery being 3.3%. From the elution pattern of PTTHactivities at steps
15 and 16, 22K-PTTH seems to be highly heterogeneous. This is not surprising, however, because such heterogeneity has been generally found in other insect peptide hor-
mones: 4K-PTTH,16) adipokinetic mone,17'18)
melanization
and
reddish
horcol-
oration hormone,19'20) periplanetin,21) and hyperglycemic hormone.22) The isolated hormonewas carefully apportioned for analyses: a small samplewasused
Prothoracicotropic
for bioassay and rechromatography, about 1fig for amino acid analysis and all the remainder for sequencing. Although sequence analysis could be done only once because of
the very limited amount available, the results clearly indicated the homogeneity of the final preparation and identified the amino-terminal
sequence of 13 amino acid residues. The sequence thus found has no significant ho-
mology with the knownpeptide or proteinous hormones including 4K-PTTH. It is of great
interest to clarify the structural relationship between 22K-PTTH and 4K-PTTH, because both have the same function, namely, to stimulate prothoracic glands to produce ecdysone, though the glands are of different insect species.
value of the protein molecule.
Purification of 22K-PTTH from a much larger number of Bombyx heads is now in progress to acquire more extensive sequence information. Acknowledgments.Weare grateful to Professor R. F.
Doolittle of the University of California for surveying amino acid sequence homology. This work was partly
supported by Grants-in-Aid for Scientific Research (Nos. 61470134 and 60105005) from the Ministry Science and Culture of Japan.
when injected
into
1)
H.
Ishizaki
and
A.
Techniques in Insects," Verlag,
New York, 1980,
2)
E.
W.
Suzuki,
"Neurohormonal
ed. by T. A. Miller, Springerpp. 244-276.
Bollenbacher
and
N.
A.
Granger,
"Comprehensive Insect Physiology Biochemistry and Pharmacology," Vol. 7, ed. byG. A. Kerkut and L. I. Gilbert, Pergamon Press, 1985, pp. 109-151.
a brainless
Bombyxpupa. This dose is comparable to the
of Education,
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