Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish

Article abstract of DOI:10.1016/j.bbrc.2017.02.009

Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N⁶-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with M_r of 47.5?K was detected. The band was lost when extract was heated at 100?°C. A similar 47.5?K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors.

Full text of DOI:10.1016/j.bbrc.2017.02.009

Biochemical and Biophysical Research Communications 485 (2017) 41e46
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Immunophotoaffinity labeling of binders of 1-methyladenine, the
oocyte maturation-inducing hormone of starfish
*
Tetsuo Toraya , Tetsuo Kida, Atsushi Kuyama, Shinjiro Matsuda, Seiichi Tanaka,
Yo Komatsu, Taro Tsurukai
Department of Bioscience and Biotechnology, Graduate School of Natural Science and Technology, Okayama University, Tsushima-naka, Kita-ku, Okayama
7
00-8530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume
meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of
starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been bio-
chemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with
immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative
maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction
Received 23 January 2017
Accepted 3 February 2017
Available online 4 February 2017
Keywords:
Immunophotoaffinity labeling
6
or its Triton X-100/EDTA extract was incubated with N -[6-(5-azido-2-nitrobenzoyl)aminohexyl]car-
boxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with anti-
1-Methyladenine
Receptor
Oocyte maturation
Starfish
body that was raised against a 1-MeAde hapten, a single band with M
r
of 47.5 K was detected. The band
ꢀ
was lost when extract was heated at 100 C. A similar 47.5 K band was detected in the crude membrane
fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and
maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder
might be a possible candidate of the starfish maturation-inducing hormone receptors.
©
2017 Elsevier Inc. All rights reserved.
1
. Introduction
probes limits its general application, immunochemical detection of
photoaffinity-labeled proteins overcomes this disadvantage [4,5].
To detect the 1-methyladenine (1-MeAde) binders in starfish oo-
cytes including putative maturation-inducing hormone (MIH) re-
ceptors, immunophotoaffinity labeling, i.e., photoaffinity labeling
combined with immunochemical detection, was attempted.
Fully-grown oocytes in the starfish ovary remain arrested at the
prophase stage of the first meiotic division. Reinitiation of meiosis
is triggered by 1-MeAde, the oocyte MIH of starfish [6], which is
produced and released by the ovarian follicle cells in response to
the gonad-stimulating substance, a peptide hormone excreted from
the radial nerve [6]. It was reported that the gonad-stimulating
substance is closely related to the vertebrate relaxin [7].
Photoaffinity labeling is a useful method to detect ligand-
binding proteins including enzymes and hormonal receptors
[1e3]. Although difficulty in radioactive synthesis of photoaffinity
6
Abbreviations:
Analog
I,
N -(6-aminohexyl)carboxamidomethyl-1-
2þ
methyladenine; ASW, modified van't Hoff's artificial seawater; CaFASW, Ca -free
artificial seawater; CBB, Coomassie Brilliant Blue R-250; EC50, 50%-effective con-
centration; EGTA, O,O’-bis(2-aminoethyl)ethyleneglycol-N,N,N ,N’-tetraacetic acid;
GVBD, germinal-vesicle breakdown; HB, homogenizing buffer or 20 mM HEPES
buffer (pH 7.4) containing
hydroxyethyl)-1-piperazinyl]ethanesulfonic acid; KLH, keyhole limpet hemocya-
nin; 1-MeAde, 1-methyladenine; MIH, maturation-inducing hormone; MPF, matu-
ration-promoting factor; PAGE, polyacrylamide gel electrophoresis; PMSF,
phenylmethanesulfonyl fluoride; Reagent I, N -[6-(5-azido-2-nitrobenzoyl)amino-
hexyl]carboxamidomethyl-1-methyladenine; Reagent II, N -[6-(3-azidobenzoyl)
0
2
5 mM EGTA and 5 mM MgCl ; HEPES, 2-[4-(2-
Upon exposure of oocytes to 1-MeAde, the maturation-
promoting factor (MPF) is activated in the cytoplasm and induces
oocyte maturation [8]. An MPF was identified as the complex of
Cdk1 kinase with cyclin B [9,10]. The involvements of pertussis
6
6
aminohexyl]carboxamidomethyl-1-methyladenine;
Reagent
III,
8-azido-1-
toxin-sensitive
G-protein,
phosphoinositide-3-kinase,
methyladenine; SDS, sodium dodecyl sulfate; Solvent A, water-saturated 2-
butanol; Solvent B, water-saturated 2-butanol containing 1% acetic acid; TLC,
thin-layer chromatography.
phosphoinositide-dependent kinase (PDK) 1, Akt (protein kinase B),
and Cdc25 phosphatase in starfish or other animal oocyte matu-
ration were suggested [11e16]. Putative 1-MeAde receptors have
*
Corresponding author.
E-mail address: toraya@cc.okayama-u.ac.jp (T. Toraya).
http://dx.doi.org/10.1016/j.bbrc.2017.02.009
006-291X/© 2017 Elsevier Inc. All rights reserved.
0

42
T. Toraya et al. / Biochemical and Biophysical Research Communications 485 (2017) 41e46
not yet been characterized biochemically, although the specific
binding of 1-MeAde to the isolated cortices of starfish oocytes was
reported independently by Yoshikuni et al. [17,18], Tadenuma et al.
water-saturated 2-butanol (Solvent A), 0.11.
To 0.5 g of this compound in 15 mL of 50%(v/v) 1,4-dioxane were
added
0.25
g
of
2-(tert-butoxycarbonyloxyimino)-2-
[
19], and by ourselves [20]. Characterization of the receptors would
help us to understand the entire signal transduction pathway from
-MeAde to MPF in starfish oocytes. For this purpose, labeling of 1-
phenylacetonitrile and 0.18 mL of triethylamine. The same
amounts of the nitrile and amine were added twice on days 2 and 4.
Six days later, the mixture was evaporated to dryness under
1
MeAde binders is a necessary step.
reduced pressure, washed with ether, and then extracted with
6
In this study, we designed and synthesized nitrene-forming
photoaffinity labeling reagents for 1-MeAde binders (Fig. 1A) and
used them for labeling the binders in starfish oocytes by a photo-
affinity labeling technique. We raised rabbit anti-1-MeAde anti-
body using a 1-MeAde derivative as hapten and used it for the
detection of the photoaffinity-labeled proteins by western blotting.
The principle of this labeling method is shown in Fig. 1B. Evidence
for the presence of a similar 1-MeAde binder in oocytes and in testis
as well as for heat stability and down-regulation of the binder are
also provided here.
water
for
N -(6-tert-butoxycarbonylaminohexyl)carbox-
amidomethyladenosine. The product was purified by silica gel
column chromatography using Solvent A. R
f
on silica gel TLC in
Solvent A, 0.62.
This compound was methylated with CH
described previously [20]. of N -(6-tert-butox-
3
I, essentially as
6
R
f
ycarbonylaminohexyl)carboxamidomethyl-1-methyladenosine on
silica gel TLC in Solvent A, 0.32. The product was hydrolyzed to
ꢀ
Analog I by heating at 95 C for 45 min in 0.5 N HCl in a sealed glass
tube. After neutralization, Analog I was purified by HPLC on Cos-
mosil C18 using 1% acetic acid (retention time, 4.6 min at a flow rate
of 2 mL/min). Its purity was established by silica gel TLC in Solvent
1
2
. Materials and methods
A (R , 0.03). H NMR (D O):
d
1.08 (m,2H), 1.15 (m,2H), 1.31 (m,2H),
f
2
1
.43 (m,2H), 2.79 (t,2H), 3.04 (t,2H), 3.80 (s,3H), 4.68 (s,2H), 8.12
(s,1H), 8.37 ppm (s,1H). UV: in nm 269 at pH 1, 273 at pH 7, and
2.1. Synthesis of Analog I
l
max
275 at pH 13.
6
N -Carboxymethyladenosine (0.55 g) [20] and 2.5 g of hexam-
ethylenediamine were allowed to react at room temperature for 2 h
in 40 mL of water at pH 4e5 with gentle stirring in the presence of
2.2. Synthesis of Reagents I and II
0
3
0
.4 g of water-soluble carbodiimide or 1-(3-dimethyaminopropyl)-
-ethylcarbodiimide. The carbodiimide was then added six times,
.1 g each time. Four days later, the reaction was terminated by
6
To N -(6-aminohexyl)carboxamidomethyladenosine (11 mg)
and 23 mg of N-5-azido-2-nitrobenzoyloxysuccinimide were
allowed to react at room temperature for 50 h in 5 mL of N,N-
adding 80 mL of water, and the supernatant was loaded onto a
florisil column (bed volume, 103 mL). After washing with water, N -
6
3
dimethylacetamide in the dark with gentle stirring. CH I (0.4 mL)
(
8
6-aminohexyl)carboxamidomethyladenosine was eluted with
0%(v/v) acetone containing 1%(v/v) acetic acid. Its purity was
established by thin-layer chromatography (TLC) on silica gel. R in
was then added, and the mixture was kept at room temperature for
47 h in the dark. N -[6-(5-azido-2-nitrobenzoyl)aminohexyl]car-
boxamidomethyl-1-methyladenosine obtained was hydrolyzed by
6
f
Fig. 1. The 1-MeAde derivatives used in this study (A) and the principle of immunophotoaffinity labeling of 1-MeAde binders in starfish oocytes (B). (A) I, 1-MeAde; II, Analog I; III
Reagent I; III , Reagent II; IV, Reagent III. (B) MA, 1-methyladenine; MA-N , photoaffinity labeling reagent containing an azide group; P, 1-MeAde binders including putative 1-MeAde
receptors; -XH, generic functional group reactive with a nitrene; -MA, anti-1-MeAde antibody; AP, alkaline phosphatase.
a
,
b
3
a

T. Toraya et al. / Biochemical and Biophysical Research Communications 485 (2017) 41e46
43
ꢀ
heating at 95 C for 30 min in 0.5 N HCl in a sealed tube. After
2.6. Preparation of oocyte crude membrane fraction and its Triton
X-100/EDTA extract
neutralization, Reagent I was purified by first TLC on silica gel 60
F
254 (20 ꢁ 20 cm) using Solvent A, second TLC on the same
adsorbent using Solvent A, and third TLC on the same adsorbent
using water-saturated 2-butanol containing 1% acetic acid (Solvent
Dejellied oocytes prepared from immature oocytes by washing
three times with CaFASW containing 2 mM EGTA (pH 6.5) were
packed by centrifugation at 120 ꢁ g for 30 s. After the addition of 2
packed volumes of homogenizing buffer (HB) containing 1 mM
phenylmethanesulfonyl fluoride (PMSF), oocytes were homoge-
nized using a Teflon homogenizer (5 strokes). The homogenates
were centrifuged at 17,000 ꢁ g for 15e30 min to remove the su-
pernatant. Ten precipitant volumes of HB containing 1 mM PMSF
were then added to the precipitate, and the suspension was
centrifuged again. This washing procedure was repeated three
times. The precipitate was suspended in a small amount of HB
B). A photosensitive band with R
f
of 0.24e0.27 in Solvent A and 0.23
in Solvent B was collected. max in water: 317 nm and ~270 nm
l
(
shoulder).
Reagent II was synthesized by the same method, except that N-
-azidobenzoyloxysuccinimide was used instead of N-5-azido-2-
3
nitrobenzoyloxysuccinimide.
2.3. Synthesis of Reagent III
ꢀ
ꢀ
8
-Bromoadenosine (100 mg) was heated at 75 C for 6 h with
containing 1 mM PMSF and stored ꢂ80 C as the oocyte crude
3
3 mg of NaN
3
in 10 mL of dimethylformamide. After dilution with
membrane fraction.
þ
water, the mixture was applied on to a Dowex 50 (H ) column.
After washing the column successively with water and 30% ethanol,
Oocyte crude membranes were re-suspended in an appropriate
amount of 5e10 mM potassium phosphate buffer (pH 8.0) con-
taining 1e2% Triton X-100, 1e5 mM EDTA, and 1 mM PMSF. After
8
-azidoadenosine was eluted with 1 N NH
silica gel TLC in Solvent B, 0.69. UV: max in nm 284 at pH 1 and
85 at pH 7 and 13. 8-azidoadenosine (40 mg) was methylated with
4 f
OH in 30% ethanol. R on
ꢀ
l
intermittent sonication for 2.5 min and shaking at 4 C for 15 min,
2
the suspension was centrifuged at 100,000 ꢁ g for 1 h. This su-
CH
3
I, essentially as described previously [20]. The mixture was
pernatant was used as the Triton X-100/EDTA extract.
diluted with water and applied on to a carboxymethyl-cellulose
þ
(
H ) column. The column was washed successively with 190 mL
2.7. Preparation and affinity purification of antibody against a 1-
of water and 190 mL of 30% ethanol, and 8-azido-1-
methyladenosine was eluted with 1 N NH OH in 30% ethanol. R
on silica gel TLC in Solvent B, 0.13. UV: max in nm 285 at pH 1 and 7
MeAde hapten
4
f
l
A 1-MeAde hapten was prepared by coupling of Analog I (5 mg)
with keyhole limpet hemocyanin (KLH) (7.7 mg) in 1.9 mL of 0.1 M
NaHCO in the presence of 2.8 mg of dimethyladipimidate$2HCl.
3
and 286 at pH 13. 8-azido-1-methyladenosine was hydrolyzed by
ꢀ
heating at 75 C for 3 h in 3.6 mL of 0.5 N HCl. After neutralization,
Reagent III was purified by HPLC on Cosmosil C18 using 2.5%
The mixture of 0.4 mL of the solution containing 1-MeAde-KLH,
0.5 mL of saline, and 1.1 mL of Freund complete adjuvant was
injected to the back of female adult rabbits. The mixture of 0.2 mL of
the 1-MeAde-KLH solution, 0.7 mL of saline, and 1.1 mL of Freund
incomplete adjuvant was used for the booster injections on days 21
and 32 after the first immunization. Whole blood was collected
seven 7 days later, and sera were obtained by a centrifuge.
methanol containing 1% acetic acid (retention time, 16.8 min at a
1
flow rate of 2 mL/min). R
f
on silica gel TLC in Solvent B, 0.30. H
NMR (D O): 3.17 (s,3H), 7.59 ppm (s,1H). UV:
2
d
lmax in nm 287 at pH
1, 293 at pH 7, and 294 at pH 13.
2.4. Analytical procedures, SDS-PAGE, and protein staining
Anti-KLH antibody was precipitated from the antiserum (3 mL)
ꢀ
The concentrations of 1-MeAde derivatives were determined by
by shaking at 4 C for 2.5 d with 18 mg of KLH in 1.5 mL of Tris-
measuring the absorbance at 270 nm [20]. UVevisible spectra were
taken on a Millton Roy Spectronic 3000 Array recording spectro-
photometer. TLC was performed on Merck silica gel 60 F254 pre-
coated glass plates using Solvent A (water-saturated 2-butanol) or
Solvent B (water-saturated 2-butanol containing 1% acetic acid).
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis
buffered saline. Anti-1-MeAde antibody was then purified by af-
finity purification using a 1-MeAde-Sepharose 4B column (bed
volume, 3 mL). The affinity-purified antibody was passed three
times through a KLH-Sepharose 4B column (bed volume, 3 mL).
2.8. Immunophotoaffinity labeling of 1-MeAde binders
(
PAGE) was carried out as described by Laemmli [21]. Protein was
stained with Coomassie Brilliant Blue R-250 (CBB). Molecular
weight markers used were SDS-7 or SDS-6H (Sigma).
Reagent I was added to 10
or its Triton X-100/EDTA extract at a concentration of 30e35
m
L of a crude membrane suspension
mM in
the dark. After 30 min at room temperature, the mixture was
ꢀ
2
.5. Animals, preparation of follicle-free oocytes, and measurement
photo-irradiated at 0 C for 2e5 min with a 250-W tungsten light
ꢀ
of hormonal activity
bulb from the distance of 20 cm. After heating at 100 C for 5 min in
the Laemmli's sample buffer, the mixtures were subjected to SDS-
PAGE. The photoaffinity-labeled proteins transferred to a PVDF
membrane were visualized by western blotting using rabbit anti-1-
MeAde antibody as a primary antibody and alkaline phosphatase-
conjugated goat anti-rabbit IgG antiserum (EY Laboratories, Inc.,
San Mateo, CA) as a secondary antibody.
The starfish Asterina pectinifera (renamed Patiria pectinifera)
collected during the breeding season near Hashirimizu, Asamushi
ꢀ
and Ushimado, Japan were kept at 14 C in laboratory aquaria
supplied with circulating artificial seawater (products from Senju
Pharmaceutical Co., Ltd., Kobe, and Rohto Pharmaceutical Co., Ltd.,
Osaka, Japan). A. pectinifera immature oocytes without follicle cells
were obtained from dissected ovaries by the transfer from Ca2þ
-
3. Results and discussion
free artificial seawater (CaFASW) [22] to modified van't Hoff's
artificial seawater (ASW) [23], washed three times with CaFASW,
and kept in ASW. The hormonal activity of 1-MeAde derivatives
was estimated from germinal-vesicle-breakdown (GVBD)-inducing
activity toward immature oocytes and expressed in % GVBD [20,23].
A 50%-effective concentration (EC50) is defined as an agonist con-
centration for inducing 50% GVBD.
3.1. Design and synthesis of photoaffinity labeling reagents
The structure-activity and the structure-affinity relationships of
1-MeAde derivatives reported so far [20,24e27] indicate the strict
specificity of putative 1-MeAde receptors for a relatively small N-1
6
substituent, whereas bulkiness and polarity of the N -substituents

4
4
T. Toraya et al. / Biochemical and Biophysical Research Communications 485 (2017) 41e46
do not so markedly affect the MIH activity. 8-MeNH-1-MeAde and
-Me N-1-MeAde serve as potent antagonists [27]. From these
at 266 nm decreased and a new broad shoulder ~320 nm increased.
Isosbestic points were observed at 303 and ~244 nm. With Reagent
III, the absorption peak at 290 nm decreased, and isosbestic points
were observed at 265 and ~247 nm. All of these results offer evi-
dence for the presence of a photosensitive azide group in Reagents I
- III.
8
2
studies, we designed and synthesized three photoaffinity labeling
reagents (Reagents I - III) for 1-MeAde binders including putative
receptors for 1-MeAde. Upon photo-irradiation, these aryl azide
derivatives of 1-MeAde would generate the super-active species
nitrenes that are expected to react with nearby functional groups of
the proteins.
3.3. GVBD-inducing activity of Reagents I-III toward starfish
oocytes
3.2. Photolysis of Reagents I - III
Reagents I-III were tested for GVBD-inducing activity toward
A. pectinifera oocytes. Since EC50 varies significantly depending
upon starfish individuals, one of the typical results is shown in
Table 1.
All the three reagents served as agonists in the induction of
GVBD, although their EC50 values were much higher than that for 1-
MeAde. Since the starfish oocytes that undergo GVBD by 1-MeAde
Reagent I underwent rapid photolysis upon irradiation of visible
light. Fig. 2A shows its spectral changes upon photo-irradiation in
an ice-water bath with a tungsten light bulb. The absorption peak at
3
17 nm due to its azidonitrobenzoyl group decreased, and new
shoulders at ~394 and ~262 nm increased with time of irradiation.
Isosbestic points were observed at 368, ~285, and ~250 nm, indi-
cating that Reagent I was photolyzed directly to another compound
without intermediates. The photolytic decomposition followed the
first-order reaction kinetics with a half-life of 2.5 min under the
conditions (Fig. 2B). Upon prolonged irradiation to ~2 h, the plot
deviated from a straight line, probably due to further decomposi-
tion of the photolytic product. Reagents II and III also underwent
rapid photolysis following the first-order kinetics upon irradiation
of UV light (data not shown). With Reagent II, the absorption peak
[23] or its analogs [20] extrude two polar bodies [23] and are
fertilizable upon insemination (elevation of fertilization membrane
and cleavage) [20], the GVBD-inducing activity may be considered
as a measure of MIH activity. It was thus concluded that all of these
reagents interact with 1-MeAde receptors and function as MIH
agonists. It is intriguing that photolyzed Reagents I - III also served
as agonists with EC50 values rather lower than those before
photolysis (data not shown), although photolytic products from
them have not yet been identified.
3.4. Immunophotoaffinity labeling of 1-MeAde binders with
membrane and soluble fractions of oocytes and testis
Crude membrane and soluble fractions of oocytes and testis
were subjected to immunophotoaffinity labeling with Reagent I. As
shown in Fig. 3A(b), a single band with M of 47.5 K was detected
r
with the oocyte crude membrane fraction, but not with the soluble
fraction. This might suggest that the 1-MeAde binder is located on
the oocyte membrane. When tested with testicular fractions, a
r
similar band with M of 47.5 K was detected with the membrane
fraction, but not with the soluble fraction. This is suggestive of the
presence of a similar 1-MeAde binder in testis. Male starfish sheds
gamete upon injection of MIH that was identified as 1-MeAde [6]
and induces spawning of sperm when added to a piece of testic-
ular tissues [23,28]. It seems reasonable to assume that female and
male starfish have similar, membrane-bound 1-MeAde receptors in
oocytes and testis, respectively.
We synthesized N-(5-azido-2-nitrobenzoyl)ethanolamine that
is related to Reagent I but lacks the 1-MeAde moiety. This reagent
and anti-1-MeAde antibody did not constitute an effective labeling
system for the 47.5 K band in the oocyte crude membrane fraction
(data not shown). It is therefore evident that the 1-MeAde moiety is
required for the immunophotoaffinity labeling of the 47.5 K band.
Other azide group-containing compounds, Reagents II and III,
were also examined for the effectiveness as a photoaffinity labeling
reagent for 1-MeAde binders using the Triton X-100 extract of
oocyte crude membranes. When these reagents were used instead
Table 1
GVBD-inducing activity of Reagents I-III.
Run no.
1
Compound
EC50 (mM)
1-MeAde
Reagent I
1-MeAde
Reagent II
1-MeAde
Reagent III
0.09
14
0.08
80
0.04
1.7
Fig. 2. Photolysis of Reagent I. Aqueous solution of Reagent I (35
mM) in a quartz
2
3
cuvette was photo-irradiated in an ice-water bath with a 250-W tungsten light bulb
from a distance of 20 cm, and UVevisible spectra were taken every 2 min (A)
UVevisible spectra (0, 2, 4, 6, 8, 10, 12, 14, and 16 min from top to bottom at 317 nm. (B)
Semi-logarithmic plot of the absorbance at 317 nm against time of photo-irradiation.

T. Toraya et al. / Biochemical and Biophysical Research Communications 485 (2017) 41e46
45
Fig. 3. Immunophotoaffinity labeling of 1-MeAde binders with membrane and soluble fractions of oocytes and testis (A) and with whole oocyte cells (B). (A, B) Appropriate amounts
of soluble and precipitant fractions of oocytes and testis (A) or oocytes (ca.100 cells) untreated (immature oocytes) or treated at room temperature with 10 M 1-MeAde for 20 min
L of HB. The mixtures were then photo-
m
(
maturing oocytes) or 60 min (mature oocytes) (B) were incubated at room temperature for 25e30 min with 30e35
m
M Reagent I in 12
m
ꢀ
irradiated in an ice-water bath for 5 min (A) or 2 min (B) with a 250-W tungsten light bulb from a distance of 20 cm. After heating at 100 C for 5 min in the Laemmli's sample buffer,
the mixtures were subjected to SDS-PAGE on 7.5% gel. Photoaffinity-labeled 1-MeAde binders were detected by western blotting, as described in the text. (a) Protein staining with
CBB. (b) Western blotting. Arrow-heads indicate the position of the 47.5 K binder.
of Reagent I and UV light was irradiated for 0.5e1 min instead of
visible light, the 47.5 K 1-MeAde binder was not detected with
Reagent III and detected only faintly with Reagent II (data not
shown). Ineffectiveness of Reagent III might be due to the absence
of an appropriate functional group in the 1-MeAde binder near the
super-active nitrene species formed from its azide group on C-8.
suggests that the 47.5 K 1-MeAde binder is proteinaceous.
Upon the preliminary incubation of the Triton X-100/EDTA
extract with 1-MeAde or Analog I, the intensity of the photoaffinity
label of the 47.5 K 1-MeAde binder was much lowered by Analog I,
3.5. Immunophotoaffinity labeling of 1-MeAde binders with whole
oocyte cells
Since the specific binding of 1-MeAde to the isolated cortices
suggested the location of the putative 1-MeAde receptors on the
surface of oocytes [17e20], immunophotoaffinity labeling was
performed with whole cells of immature, maturing, and mature
oocytes. When immature and maturing oocytes were photoaffinity-
labeled with Reagent I, followed by western blotting with anti-1-
MeAde antibody, a single band with M
r
of 47.5 K was detected
(Fig. 3B(b)). In contrast, this band was very faint with mature oo-
cytes, suggesting the internalization of the 1-MeAde binder upon
oocyte maturation. This might be as expected if this 1-MeAde
binder is a cell-surface receptor for MIH and undergoes down-
regulation in the hormonal signal transduction.
3.6. Extraction and some properties of the 47.5 K 1-MeAde binder
Fig. 4. Extraction from oocyte membrane fractions and heat stability of the 1-MeAde
binders (A,B) and inhibition of photoaffinity labeling by Analog I (C). Crude membrane
fraction (1 mL) washed with HB was extracted by sonication for 2.5 min successively
with 1% Triton X-100 and 1% Triton X-100 containing 1 mM EDTA, 1 mL each. Each
extract was separated by ultracentrifugation at 100,000ꢁg, and the residual precipitate
was suspended in the same volume of HB. They were subjected to SDS-PAGE, protein
staining with CBB (A), and immunophotoaffinity labeling (B), as described in the text.
Extraction of the 47.5 K 1-MeAde binder from the oocyte
membrane fraction was attempted using a Triton X-100 with or
without EDTA. As shown in Fig. 4B, the addition of 1 mM EDTA to 1%
Triton X-100 markedly increased the efficiency of extraction,
although the significant amount of 47.5 K 1-MeAde binder still
remained in the residual precipitate.
ꢀ
The Triton X-100/EDTA extract heated at 100 C for 10 min was analyzed by immu-
nophotoaffinity labeling (C). Immunophotoaffinity labeling with the Triton X-100/
EDTA extract was also carried out after pre-incubation with 1 mM 1-MeAde or Analog I
at room temperature for 30 min (C). Arrow-heads indicate the position of the 47.5 K
binder.
When the Triton/EDTA extract was subjected to immunopho-
ꢀ
toaffinity labeling after heat treatment at 100 C for 10 min, the
r
band with M of 47.5 K was no more detected (Fig. 4C). This result

46
T. Toraya et al. / Biochemical and Biophysical Research Communications 485 (2017) 41e46
but almost not by 1-MeAde (Fig. 4C). This might suggest that
Analog I is bound to the binder in more irreversible manner than 1-
MeAde itself.
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[
[
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11] F. Shilling, K. Chiba, M. Hoshi, T. Kishimoto, L.A. Jaffe, Pertussis toxin inhibits
In summary, we designed and synthesized photoaffinity label-
ing reagents for 1-MeAde binders of starfish. For immunochemical
detection of photoaffinity-labeled 1-MeAde binders, rabbit anti-1-
MeAde antibody was raised. A 47.5 K 1-MeAde binder was detec-
ted both in oocytes and testis crude membrane fractions by
immunophotoaffinity labeling. This heat-labile binder was labeled
with whole oocytes cells as well and underwent down regulation
after maturation. The binder might be a possible candidate of MIH
receptors of starfish oocytes, a possibility that should be tested in
the near future.
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(
1989) 605e608.
12] S. Hehl, B. Stoyanov, W. Oehrl, R. Sch o€ nherr, R. Wetzker, S.H. Heinemann,
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[
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[
[
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
[
18] M. Yoshikuni, K. Ishikawa, M. Isobe, T. Goto, Y. Nagahama, Synergistic actions
of disulfide-reducing agents on 1-methyladenine-induced oocyte maturation
in starfish, Dev. Biol. 128 (1988) 236e239.
[
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We thank Tadamasa Ohmiya, Tomoko Tanabe, Yuri Saito, and
Masatoyo Yamamoto for their helpful discussions.
[
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