VIII-Ⅱ-1. Project Research
Project 11PR11

Project Research on the Abnormal Aggregation of Proteins by Post-translational Modifications, andStudy of Repair Mechanism

N. Fujii

Research Reactor Institute, Kyoto University
Objectives and Allotted Research Subjects:
The aim of this project research is to elucidate thecorrelation between the change of the protein structure
induced by various post-translational modifications
with UV irradiation, gamma-irradiation, aging andprotein function. We also investigate the repair mechanism for the damaged protein by irradiation. This
research program has started in 2011. In this year, the
7 research subjects were carried out. The allotted research subjects (ARS) are as follows;
ARS-1: Structural change of eye lens protein(crystallin) in solution. (M. Sugiyama, Y. Ueki, N. Fujii
and N. Fujii)

ARS-2: Evolutinal Consideration of
D-Aspartyl
Endopeptidase. (T. Kinouchi and N. Fujii)
ARS-3: Damage to biological molecules induced byionizing radiation and biological defense mechanisms
provided by radical scavengers. (T. Saito and N. Fujii)
ARS-4: Effect of structural alteration of asparagine
residue in the Prion peptide fragment (106-126) on theamyloid fiber formation. (Y. Sadakane and N. Fujii)
ARS-5:Analysis of environmental stress-related hearing loss in the mice. (N. Ohgami and N. Fujii)
ARS-6: Effect of gamma-ray irradiation on the activity of tyrptophanase and tryptophan synthase.
(A. Shimada, N. Fujii and T. Saito)

ARS-3: Saito et al. showed that beta-carotene does notaffect the linolenic acid peroxidation reaction induced by
gamma irradiation, suggesting that carotenoid pigments
affect radical reactions that proceed after the lipid peroxidation reaction in the biological defense mechanism invivo.
ARS-4: Sadakane et al. analyzed the effect of structural
alterations of asparagine residue on the amyloid fiber
formation by measuring thioflavin fluorescence and observing the solution viscosity, and revealed that the viscosity of peptide solution containing D-form aspartic
acid increased. types of damage to biological lipids invivo.
ARS-5: Ohgami et al. showed that partial impairment of
c-Ret causes age-related hearing loss involving
neurodegeneration of spiral ganglion neurons in mice.
Neurodegenerative disorders usually involve impairments
of protein including aggregation and oxidative stress. We
are investigating whether hearing loss involves aggregation of a specific protein in inner ears.
ARS-6: This research aims to investigate how the activity of tryptophanase (TPase) and tryptophan synthase(TSase) changes when a low dose of γ-rays is administered to them. We first investigate the influence of it
about tryptophanase’s activity.
ARS-7: Fujii et al. found that the simultaneous stereo
inversion and isomerization at the Asp 4 residue occur inbeta B2-crystallin from the cataractous lenses of elderly
donors.

ARS-7: Simultaneous stereoinversion and isomerization
at the Asp-4 residue in beta B2-crystallin from thecataractous lenses. (N. Fujii, T. Kawaguchi, H. Sasaki
and N. Fujii)

Main Results and Contents of This Project
et.al. investigated a structure ofB2-crystallin in an aqueous solution . As a result, it
was revealed that the structure of B2-crystallin is different from that in crystal.

ARS-1: Sugiyama

ARS-2: Kinouchi and Fujii examined the distribution of
D-aspartyl-endopeptidase (DAEP) in animals and microorganisms. As a result DAEP activity was detected infishes, amphibians and birds, but was not detectablein archaea, prokaryotes, yeast and nematode. Interestingly high DAEP activity in African clawed frog
(Xenopus laevis) was remarkable in testis and unfertilized eggs. It is suggested that a primitive physiological function of DAEP is associated with fertilization
and/or oocyte maturation.

採択課題番号 23P11（京大原子炉）藤井紀子

放射線や紫外線照射によるタンパク質の異常凝集とその防御及び修復機構に関する研究

プロジェクトPR11-1

Structural Change of Eye Lens Protein (crystalline) in Solution

M. Sugiyama, Y. Ueki, N. Fuji and N. Fuji
Research Reactor Institute, Kyoto University
INTRODUCTION: Crystallin is a main componentof eye lens proteins. There are three kinds of crystallins
in human eye lens, α-crystallin, β-crystallin andγ-crystallin. α-Crystallin, the largerst proten among three
crystallins (Mw is ca 800kDa), belongs to a family ofheat shock proteins and it therefore works as a chaperon
to repair the other proteins. On the other hands, the functions of β- and γ-crystallins have not been clarified yet.
To understand their roles and functions in eye lens, as thefirst step, we begin to clarify the structure of β-crystallinin an aqueous solution.
It is well-known that protein structure can be determined with high resolution by single crystal X-ray diffraction method. However, it is difficult to reveal theprotein structure in the an aqueous solution with thismethod. Therefore, we employed a small-angle neutron
scattering (SANS) method for this propose, the clarification of protein structure in an aqueous solution. Even
though Small-Angle Scattering has lower spatial resolution than that of single crystal diffraction, it does not require “crystal” as a sample and moreover can observe theparticle structure in a solution. In addition, neutron is a
soft probe for the sample, not to make less radiation
damage on the protein. However, it is difficult to reveal
the detailed structure by ordinary SANS data analysis
method, which gives us approximative size and/or shapeof a particle. Therefore, we are developing a new analysis method utilizing a RMC technique.

Fig. 1. Structure(PDB:1ytq) .

ofB2-crystallin

Figure 2 shows a SANS profile of B2-crystallin in an
aqueous solution and the simulated one. The simulated
SANS profile does not reproduce the experimental one.
The gyration radius calculated from the experimental
one is 20.8 Å but that from the simulated one is23.5Å. This clearly suggested that the distance
between two domains changes in an aqueous
solution, compared with that in the crystal.
Now, we are trying to reveal the distance in aqueous
solution by RMC-coupled analysis.

EXPERIMENTS: Human B2-crystallin expressedby E.Coli was used as a sample. The concentrations ofsamples were tuned to be 3.0 mg/ml and the solvent was
20 mM Tris/HCl (pH 7.8) + 150 mM NaCl.The used
SANS data were observed with SANS-U installed at
JRR-3 in Tokai.
ANALYIS: The structure of B2-crystallin (1ytq) is

shown in Fig.1. This protein has two domains with thesize of 10Å apart from 24Å. Firstly, we have to develop the SANS simulation method; the simulation code
makes the SANS profile from the single crystal structure data. By cubic-dividing method, we successfully
obtain a SANS profile from PDB data.

採択課題番号 23P11-1（京大・原子炉）杉山正明

Fig. 2. SANS profile of -crystallins (opencircles) and simulated one with PDB data(line).

Crystallin 複合体の会合様態の研究

プロジェクトPR11-2

Evolutional Consideration of D-Aspartyl Endopeptidase

T. Kinouchi and N. Fujii
Research Reactor Institute, Kyoto University
INTRODUCTION: We have been characterizing
mammalian D-aspartyl endopeptidase (DAEP) [1-3].
Significant features of DAEP are as follows: DAEP
forms a large ring-like complex structure, such as the
AAA+ protease [2], and stereoselectively recognizes anddegrades its substrate at the internal D--aspartate (Asp)residue in an ATP-independent manner [3]. As can beseen from many reports, D-Asp is detected in abnormallyaggregated proteins causing age-related diseases (i.e.,
cataract, prion disease and Alzheimer's disease), and it isstrongly suggested that formation of D-isomer of Asp
residue in human proteins is potentially noxious for thenormal protein turnover in our cell. Therefore DAEP
seems to physiologically serve as a kind of quality-control system against the misfolded D-Asp
containg-protein and to consequently maintain the protein homeostasis in high body-temperature and long life
span animals such as mammals. On the other hand, a
distribution of DAEP in living things has not been examined, and the results would be highly informative forthe further functional characterization and the discovery
of a primitive homolog of DAEP. We therefore started
examining the distribution of DAEP in various animals
and microorganisms.
EXPERIENTS: Materials>Archaea: Thermoplasma
acidophilum, Archaeoglobus fulgidus, Pyrococcus
horikoshii, and Thermococcus celericrescens were purchased from NBRC (NITE Biological Resource Center).
Prokaryotes: E.coli, Thermus thermophiles (was kindly
gifted by Dr. Kunio Miki, Kyoto University), andDeinococcus radiodurans (was kindly gifted by Dr.
Takeshi Saito, KURRI). Eukaryotes: yeast (Saccharomyces cerevisiae), nematoda (Caenorhabditis elegans),
fishes: medaka (Oryzias latipes, was purchased from a
local petshop), amphibians: African clawed frog
(Xenopus laevis, was kindly gifted by Dr. Yuichi Tsuchiya, Toho University), birds: chickens (Gallus gallus
domesticus, purchased from a local slaughterhouse).
Screening of DAEP activity>We developed an assay system for DAEP activity using the synthetic D-Asp containing
substrate,
Succinyl-D-Asparticacid
-(4-methyl-coumaryl-7-amide) (Suc-D-Asp-MCA) [1].
Homogenate of the above biological materials was mixed
and incubated with 0.1 mM Suc-D-Asp-MCA and theassay buffer (10 mM Tris-HCl (pH 8.5), 200 mM NaCl, 3
mM MnCl2) at optimum temperature for each. The fluorescence of aminomethylcoumarin liberated from
Suc-D-Asp-MCA by DAEP is measured at ex = 380 nm
and em = 460 nm.
RESULTS & DISCUSSION: DAEP activity was detected in fishes, amphibians and birds, but was not detectable in archaea, prokaryotes, yeast and nematode,
which were examined in this study. As we reported,mammalian liver was shown to have the highest DAEP
activity among tissues (fig. 1) [1]. Even in chickens,
DAEP activity was distributed in their livers. On theother hand, the tissue distribution of DAEP activity in
African clawed frog, Xenopus laevis was quite different
from that in mammals and birds. As shown in fig. 1,
high DAEP activity in Xenopus laevis, was remarkable intestis and unfertilized eggs, but was imperceptible in liver. Since Xenopus laevis is an animal ranking below
mammals and birds in the viewpoint of evolutionary biology, a primitive physiological function of DAEP is
considered to be associated with fertilization and/or oocyte maturation. Further analyses of Xenopus DAEP
might elucidate its essential function.

Fig. 1. DAEP activity in various tissues of rabbit and
Xenopus laevis.
Y-axis indicates specific activity of DAEP (% ofmaximum). Closed and open bars represent rabbit
and Xenopus laevis, respectively.
REFERENCES:[1] T. Kinouchi et al., Biochem. Biophys. Res. Commun.,
314 (2004) 730-736.
[2] T. Kinouchi et al., Chem. Biodivers., 7 (2010)
1403-1407.
[3] T. Kinouchi et al., J. Chromat. B., 879 (2011)3349-3352.

採択課題番号 23P11-2
D-アスパラギン酸含有蛋白質に特異的な分解酵素の研究（京大・原子炉）木野内忠稔、藤井紀子

プロジェクトPR11-3 Damage to Biological Molecules Induced by Ionizing Radiation and Biological Defense

Mechanisms Provided by Radical Scavengers
T. Saito and N. Fujii
Research Reactor Institute, Kyoto University
INTRODUCTION: Some bacteria exhibit very highresistance to ionizing radiation [1]. A common feature ofthese bacteria is that they contain red carotenoid pigments [1, 2, 3]. Because colorless mutants of these radioresistant bacteria are more sensitive to gamma irradiation than wild types, the red carotenoid pigments arethought to contribute to the bacteria’s defense mechanisms against ionizing radiation [1]. It is well known that
biological effects induced by low-LET ionizing radiation
are caused mainly by radicals generated by radiolysis.
Carotenoid pigments exhibit high radical scavenging
activity and are localized within the lipids of the cell surfaces in prokaryotes. These facts indicate that red carotenoid pigments in radioresistant bacteria must defend
the lipids of the cell surfaces of these bacteria against
ionizing radiation.
When considering the biological defense mechanismof these radioresistant bacteria against ionizing radiation,
it is important to elucidate the effects of carotenoid pigment as regards damage to biological molecules. We
have reported that linolenic acid is oxidatively degradedand peroxidized by gamma irradiation in adose-dependent manner [4]. In this study, we analyzed
the effect of beta-carotene, which is a typical carotenoid
pigment, on the peroxidation of linolenic acid by gamma
irradiation.
EXPERIMENTS: Sample Preparation: Linolenic acidwas dissolved in benzene at a final concentration of 5.0 x
10-1 M and beta-carotene was added at final concentrations of 5.0 x 10-8 to 5.0 x 10-4 M. Gamma Irradiation:
The prepared solutions were irradiated with 60Co gamma
rays at a dose of 30 kGy and a dose rate of 25 kGy/h.
Linolenic Acid Peroxidation Assay: A modified versionof the method described by Pryor and Castle was used[5]. The gamma-irradiated sample was diluted 600 times
with benzene, and then 5 ml of the diluted solution was
evaporated in vacuo. The residue was dissolved in 5 mlof n-hexane, and the average absorbance from 230-236
nm of this solution, which was derived from the generated conjugated diene, was measured. In this study, thelevel of linolenic acid peroxidation was evaluated by theamount of generated conjugated diene.
RESULTS: Under this experimental condition,beta-carotene had no significant effect on the peroxidation reaction of linolenic acid induced by gamma irradiation. We have previously reported that beta-carotene affects the oxidative degradation reaction of linolenic acidinduced by gamma irradiation using an analysis in which
the level of the oxidative degradation of linolenic acidwas evaluated by the amount of generated malondialdehyde [6]. The lipid damage induced by radicals begins
with a hydrogen abstraction reaction followed by a peroxidation reaction. Then there is a degradation reactionthat includes a radical reaction. The above reveals thatbeta-carotene does not affect the linolenic acid peroxidation reaction during the initial radical reaction, which hasa high reaction rate, but affects the linolenic acid degradation reaction that proceeds after the peroxidation reaction in the linolenic acid damage process induced by
gamma irradiation.

Fig. 1. The effect of beta-carotene on the amount of gen-erated conjugated diene induced by the gamma irradiation of linolenic acid at a dose of 30 kGy. The horizontal
axis shows the beta-carotene concentration, and the vertical axis shows the 230-236 nm average absorbance,
which reveals the relative amount of generated conjugated diene. Each value is expressed as an average ±S.D. of three separate experiments.
REFERENCES:[1] T. Saito, Viva Origino, 30 (2007) 85-92.
[2] T. Saito et al., Arch. Microbiol., 162, (1994) 414-421.
[3] T. Saito et al., Microbios, 95, (1998) 79-90.
[4] T. Saito and N. Fujii, KURRI Progress Report 2010,(2011) 105-105.
[5] W. A. Pryor and L. Castle, Meth. Enzymol., 105,
(1984) 293-299.
[6] T. Saito and N. Fujii, KURRI Progress Report 2009,(2010) 137-137.

採択課題番号 23P11-3 放射線照射による生体分子の損傷とラジカルスカベンジャー
による生体防御機構（京大・原子炉）齊藤 毅、藤井 紀子

プロジェクトEffect of 6tructual $lteration of $sparagine 5esidue in the Prion 3eptide )ragment(106-126) on the $myloid )iber )ormation

Y. Sadakane and N. Fujii1
Department of Pharmaceutical Sciences, Suzuka University of Medical Science
1
Research Reactor Institute, Kyoto University
INTRODUCTION: The native L–asparagine (Asn) residue in the peptide and protein was converted to theL–succinimide intermediate, and it is quickly hydrolyzed
and produced the mixture containing L–Asp, L–isoAsp,
D-Asp and D-isoAsp residues (Fig. 1). The alternation of
aspartic acid (Asp) or Asn residue also has been found in
the key proteins related to neuronal disease such as prion
protein (PrP). The deamidation of Asn residue in
PrP106-126 has been reported to affect its channel activity
in planar lipid bilayer. In this study, we investigated theeffect of such structural alteration of the PrP peptide on
the amyloid fiber formation by the thioflavin fluorescentmeasurement and the solution viscosity observation.
O

H
O

H

O
NH 2
H
N

H N

N
H

O

N
H

R

αL-AsnαL-Asp

OH
H
N

N
H
O

R

O

R

R

H

O

L-succinimidyl

βL-Asp

N
H
OH

N
H
O
O

O

H

αD-Asp

10
8
6
4
2
0
N

αL βL αD βD

Fig. 2 Effect of structural alteration of asparagine residue
on the thioflavin fluorescent intensity after 37 oC incubation for 5days. N, asparagine; αL, βL, αD, and βD, aspartic acid residues.

N
H

H N
N
H

OH
H
N

thioflavin added in the peptide solution after incubating
at 37 oC for 5 days were measured by fluorescent micro-plate reader (Fig. 2). The intensities of five peptide
solution were shown no difference each other. The photograph of upside-down tubes containing peptide solutionthat had been incubated at 37 oC for a week was shown(Fig. 3). The peptide solutions bearing D-form Asp wereleaved in the bottom of tubes. This result shows that theviscosity of D-form Asp-containing peptide solution increase in comparison with that of the others.

Relative fluorescent intensity

PR11-4

O

R

R

O

O

D-succinimidyl

H

R
N
H
OH

βD-AspN
HO

N αL βL αD βD

Fig. 1 The asparagine residue generates four aspartic acids which bear the beta–linkage bond and stereo-inverted
D-form.
EXPERIMENTS: The original and four peptide isomers,(L-Asn108, L -Asp108, L-isoAsp108, D-Asp108, D-isoAsp108,
which were represented by N, αL, βL, αD, and βD in thisstudy) corresponding to the PrP106-126 sequence(KT108NMKH MAGAAAAGAVVGGLG) were preparedby Fmoc chemistry [1]. The thioflavin fluorescent intensity was measured in the peptide solution which had been
incubated at 37 oC for 5 days. The viscosity of peptide
solution was observed by using microtubes (0.2 mL vol.).
The peptide solution was incubated at 37 oC for a week
in the tubes, then the tubes were turned upside down, andthe solution state was observed.
RESULTS:The

relative

採択課題番号 23P11-4

fluorescentintensityofFig. 3 Effect of structural alteration of asparazine residue
on the viscosity of peptide solution after 37 oC incubationfor 7 days. N, asparagine; αL, βL, αD, and βD, aspartic
acid residues.
REFERENCE:[1] Y. Sadakane, K. Konoha, M. Kawahara, and K.
Nakagomi, Chem. & Biodivers. 7 (2010)1371-1379.

タンパク質中のアスパラギン酸残基の異性化と機能変化に関する研究（鈴鹿医療科学大学・薬学部）定金 豊、（京大・原子炉）藤井 紀子

プロジェクトPR11-5

Analysis of Environmental Stress-related Hearing Loss in MiceN. Ohgami and N. Fujii1
Units of Environmental Health Sciences, Department of
Biomedical Sciences, College of Life and Health Sciences, Chubu University.
1
Research Reactor Institute, Kyoto University
INTRODUCTION: Hearing level is affected by genetic,
aging and environmental factors [1,2]. About 120 million
people worldwide suffer from congenital (early-onset)
hearing loss. Thirty percent of them have syndromic
hearing loss and the remaining seventy percent havenon-syndromic hearing loss. In addition, a large numberof aged people worldwide suffer from age-related(late-onset) hearing loss [1,2]. These hearing losses havebeen generally categorized as distinct diseases due todifferent pathogeneses.
c-Ret encodes a receptor-tyrosine kinase. Glial cellline-derived neurotrophic factor (GDNF) is one of theligands for c-Ret [3]. GDNF exerts its effect on target
cells by binding to a glycosyl phosphatidylinositol(GPI)-anchored cell surface protein (GFRα1), which thenallows the receptor tyrosine kinase c-Ret to form a multi-subunit signaling complex. Formation of this complex
results in c-Ret autophosphorylation and a cascade of
intracellular signaling to control cell survival [3]. Tyrosine 1062 (Y1062) in c-Ret is one of the most crucial
autophosphorylation sites for its kinase activation and is amulti-docking site for several signaling molecules including Shc (Src Homology 2 domain-Containing transforming protein 1), a transmitter for c-Ret signaling [3].
We have recently shown that complete impairment of
Y1062-phoshorylation in c-Ret causes congenital deafness with HSCR in homozygous c-Ret Y1062F knock-in
mice (c-Ret-KIY1062F/Y1062F-mice) [4]. On the other hand,heterozygous c-Ret Y1062F knock-in mice(c-Ret-KIY1062F/+-mice) have been shown to have no
HSCR-linked phenotype [3] except for hearing levels. In
this study, we used c-Ret-KIY1062F/+-mice in order to analyze whether partial impairment of Y1062-phoshorylationin c-Ret causes age-related hearing loss.
**********************************************
EXPERIMENTS: c-Ret-KIY1062F/+-mice used in thisstudy were previously reported [3]. All experiments wereauthorized by the Institutional Animal Care and Use
Committee in Chubu University (approval number:
2210038) and the Institutional Recombinant DNA Experiment Committee in Chubu University (approval

number: 06-01) and followed the Japanese Government
Regulations for Animal Experiments. Hearing levels inmice were measured by auditory brainstem responses(ABR) [4]. Inner ears were analyzed by light microscopy
and scanning- and transmission electron microscopy [4].
Preparation of tissues for transmission electron microscopy basically followed the previous method [4].
**********************************************RESULTS:
PartialimpairmentofY1062-phosphorylation in c-Ret showed acceleration ofage-related hearing loss in heterozygous c-Ret Y1062F
knock-in mice (c-Ret-KIY1062F/+-mice) [5]. The hearing
loss involved late-onset neurodegeneration of spiral ganglion neurons (SGNs) in c-Ret-KIY1062F/+-mice [5]. Morphological abnormalities in inner- and outer-hair cells
and the stria vascularis in c-Ret-KIY1062F/+-mice werehardly detected under scanning electron microscopy andlight microscopy [5]. We further performed detailed
morphologicalanalyses
of
SGNs
from
c-Ret-KIY1062F/+-mice by transmission electron microscopy [4]. Aged c-Ret-KIY1062F/+-mice showed gaps between
SGNs and Schwann cells compared to those in littermate
WT mice [5]. Nuclei of SGNs from aged
c-Ret-KIY1062F/+-mice were obviously shrunken comparedwith those from littermate WT mice [5]. The nuclei of
SGNs from aged c-Ret-KIY1062F/+-mice exhibited discontinuous nuclear membranes, while those from littermate
WT mice had intact bilayer membranes of nuclei [5].
Degeneration at the auditory nerve fiber was observed inaged c-Ret-KIY1062F/+-mice [5]. Thus, our results suggestthat partial impairment of c-Ret causes age-related hearing loss involving neurodegeneration of SGNs in mice[4]. Neurodegenerative disorders usually involve impairments of protein including aggregation. Further study
is needed to investigate whether hearing loss involves
protein aggregation.
**********************************************REFERENCES:[1] S. D. Brown et al., Nat. Rev. Genet., (2008) 9,
277-290.
[2] Lalwani, A. K., Gürtler, N., CURRENT Diagnosis& Treatment in Otolaryngology—Head & Neck Surgery, second edition. McGraw-Hill, New York, (2008)
pp. 683-704.
[3] M. Jijiwa et al., Mol. Cell. Biol., (2004) 24,
8026-8036.
[4] N. Ohgami et al., Proc. Natl. Acad. Sci. USA.,(2010) 107, 13051-13056.
[5] N. Ohgami et al., Neurobiol. Aging., (2012) 33,
626.e25-626.e34.

採択課題番号 23P11-5 騒音ストレスによる内耳タンパク質中のアスパラギン酸残基の異性化の解析 プロジェクト（中部大学）大神信孝（京大・原子炉）藤井紀子

PR11-6 Effect of γ-Ray Irradiaiton on the Activity of Tyrptophanase and Tryptophan Syntase
A. Shimada, N. Fujii1 and T. Saito1
Sustainable Environmental Studies, Graduate School ofLife and Environment Sciences, University of Tsukuba
1
Research Reactor Institute, Kyoto University
INTRODUCTION: γ-ray-irradiated enzyme reduces its
activity because γ-ray irradiation against the enzyme
causes drastic tertiary conformational change with irreversible denaturation. When the enzyme is administrated
at lower dose, the activity should make minute change
responsive to small conformational change. Currently,
we have just few data on the activity in low dose. We
have presented many researches about interesting tryptophanase’s activity on D-tryptophan in the past. So weare quite familiar with tryptophanase. Whereas tryptophan synthase is structurally different from tryptophanase, both enzymes have similar activity. Thereby it
is of great interest to compare tryptophanase’s activity
with tryptophan synthase’s one. The present study aims
to investigate how tryptophanase (TPase) and tryptophan
synthase (TSase) change in their activities when theyreceive a low radiation dose. In this study, we first investigate about tryptophanase’s activity. Tryptophanase, an
enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the degradation of
L-tryptophan into indole, pyruvate and ammonia throughα,β-elimination mechanism, having no activity on
D-tryptophan (D-Trp). Although tryptophanase is widely
known as one of enzymes with strict stereospecificity, it
becomes active for D-Trp in a highly concentrated diammoniumhydrogen phosphate solution. This reaction
has never been reported before. This reaction activity,
however, is very low. Activity on D-Trp has only 0.7 %of one of L-Trp at 37 ℃. We investigate here whether it
is possible to elevate the activity by use of a low dose of
γ-rays.
EXPERIMENTAL: Apotryptophanase was purchasedfrom Sigma Chem. Co., prepared to a concentration of
200 μg/mℓ in 100 mM potassium phosphate buffer solution with a 20 % saturation concentration of diammoniumhydrogen phosphate (DAP) and 380 mM pyridoxal
5ʹ-phosphate. The enzyme was exposed to γ-rays, forwhich a cobalt-60 source was used with a dose rate of 3
Gy/sec. Tryptophanase was irradiated at doses of 30 –
5400 Gy. Here we wrote about γ-ray-irradiated tryptophanase as γ-TPase. The activity of TPase and γ-TPase
was assayed against D-Trp. Reaction mixture was composed of 20 % saturation DAP, 380 mM pyridoxal
5'-phosphate, 245 μM D-tryptophan and 200 μg/mℓ

択課題番号 23P11-6

TPase or γ-TPase (pH 7.8). Reaction was performed at
37 °C for 4 h. The reaction was stopped by adding
n-butanol, vigorously mixed, and then immediately centrifuged at approximately 1,000 g for 10 min. After thecentrifugation, the supernatant including indole released
from tryptohan degradation was extracted, colored with
Ehrlich’s reagent for 30 min at 60 °C, and then its
amount was spectroscopically detected at 570 nm to beconverted into concentration from calibration curve. The
activity on γ-TPase was compared with that of TPase to
analyze how a low irradiation of γ-ray influenced on
tryptophanase’s activity. Unless otherwise stated, reagents were obtained from Wako Pure Chem. Co. Ltd. All
chemicals were reagent grade. All glasswares were
washed by soaking more than 3 days in a special detergent, CLEAN 99CL (Clean Chemical Co. Ltd.), thoroughly rinsed in deionized and distilled water, and thendried in an oven.
RESULTS AND DISCUSSION: As generally-accepted
notion, tryptophanase’s activity decreased in response toincreasing γ-ray dose from 600 to some 2000 Gy. Its
reduction rate was slowed down over 2000 Gy, almost
stopping between 4000 – 5400 Gy. Tryptophanase wassusceptible to γ-ray in the same manner as other enzymes
and protein. However, we observed tryptophanase’s reaction was different from this result between 0 and 600
Gy. Prior to experiment, we forecasted the activity of
γ-TPase was far below that of TPase there. However,there was either slightly below or little difference from
TPase’s activity between them against our expectation.
In some cases, we could observe that γ-TPase’s activity
reached several percent above TPase’s one. We interpret
γ-TPase’s activity in γ-ray irradiation between 0 and 600
Gy as follows. Protein, along with enzyme, is fragile toradioactive ray. Enzyme is especially sensitive to it because its active site needs very precise tertiary structure.
When the enzyme is exposed to massive radiation exceeding some limit, the enzyme’s stereostructure is broken to be inactivated or denatured. However, when radiation dose within the limit is administered to the enzyme,
it has a small effect on the enzyme due to its radioresistance. It goes without saying that γ-ray almost always
has wrong effect on the enzyme. However, there may be
the remotest chance that it gives positive effect on the
enzyme activity. It will thus become important to analyze
the activity in detail when the enzyme is γ-irradiated at
low dose of ~ 100 grays. We predict that tertiary structural change is probably responsible for this effect, andalso we are now searching the sophisticated ways to detect it. We will make it clear in the future study.

ガンマー線を照射したトリプトファンシンターゼの立体選択性の変化（筑波大・生命環境系）島田秋彦,(京大・原子炉) 藤井紀子，齋藤 毅

プロジェクトPR11-7 Simultaneous Stereoinversion and Isomerization at the Asp-4 Residue in B2-Crystallinfrom the Aged Human Eye Lenses
N. Fujii, N. Fujii, T. Kawaguchi and *H. Sasaki
Research Reactor Institute, Kyoto University, Kanazawa
Medical University*
INTRODUCTION: A cataract, which is the most common age-related disease, is caused by clouding of the eyelens that may lead to a partial or total loss of vision. The
mechanism of cataract development is not well understood. However, it is thought that eye lens proteins of acataract are abnormally aggregated, resulting in clumpingthat scatters the light and interferes with focusing on theretina. Human lens proteins are mainly composed fromthe -, -, and -crystallin superfamily of proteins. The
overall structure, stability and short-range interactions ofthese proteins are thought to contribute to the transparent
properties of the lens. Because the lens crystallins are
long-lived proteins, they undergo various posttranslational modifications including isomerization, inversion,
deamidation, oxidation, glycation and truncation. These
posttranslational modifications may lead to age-related
cataract. Previous studies concerning the posttranslational modification of -crystallin have been limited to analyzing the deamidation of asparagine (Asn) or glutamine(Gln) residues, and oxidation of methionine and tryptophan residues [1]. Here, we demonstrate for the first timestereoinversion and isomerization of the Asp residues at
the N- terminal regions in B2-crystallin isolated fromelderly individuals [2].
EXPERIMENTS: Lens samples from elderly individuals were homogenized and the sample was subjected tosize exclusion chromatography to obtain the -crystallin.
Subsequently, -crystallin was further fractionated into
B2-crystallin and other -crystallins by reverse-phasehigh-performance liquid chromatography (RP-HPLC)
using a C4 column. The B2-crystallin was digested withtrypsin and the resulting peptides were separated by
RP-HPLC using a C18 column. The tryptic peptides ofB2-crystallin were identified using a matrix-assisted
laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOFMS) (AXIMA-TOF2; Shimadzu,
Kyoto, Japan). The peptides were hydrolyzed and to form
diastereoisomers using o-phthalaldehyde (OPA) andn-tert-butyloxycarbonyl-L-cysteine (Boc-L-cys). The D/Lratio of the amino acids was determined using RP-HPLC.
RESULTS: We found that the aspartyl (Asp) residue at
採択課題番号 23P11-7（京大・原子炉）藤井position 4 of B2-crystallin in the lenses of elderly human individuals undergoes a significant degree of inversion and isomerization to the biologically uncommon
residue D--Asp. Surprisingly, the D/L ratio of -Asp at
position 4 in B2-crystallin from elderly donors (67-77
year-old) was 0.88-3.21. A D/L ratio of amino acids
greater than 1.0 is defined as an inversion of configuration from the L- to the D-form, rather than a racemization.
DISCUSSION: Usually, the inversion proceeds withdifficulty under physiological conditions, however,
D-Asp formation occurs via a succinimide when theneighboring amino acid of the Asp residue has a small
side chain, such as glycine, alanine or serine. The residue
neighboring Asp-4 of B2-crystallin is histidine, which
has a bulky side chain, suggesting the unusual D/L ratioof Asp-4 is not influenced by the adjacent residue. Rather,
inversion of Asp-4 in B2-crystallin may proceed because of the chiral environment around this residue that
promotes inversion of the L- to the D-form. Carver et al.
clearly showed that the N- and C-terminal extensions ofbovine B2-crystallin are largely unstructured. These
extensions were shown to be accessible to solvent andpossess significantly greater flexibility compared withthe domain core of the protein [3]. Thus, the structural
data supports our hypothesis that the highly flexible region around Asp-4 could promote formation of thesuccinimide followed by inversion and isomerization.
Inversion of these Asp residues directly affects thehigher order structure of the protein. Hence, this modification may change crystallin-crystallin interactions anddisrupt the function of crystallins in the lens.
REFERENCES:[1] Bloemendal, H., de Jong, W., Jaenicke, R., Lubsen, N.
H., Slingsby, C .and Tardieu, A. Ageing and vision:
structure, stability and function of lens crystallins. Prog.
Biophys. Mol. Biol. 86, 407-485 (2004)[2] Fujii, N., Kawaguchi, T., Sasaki, H. and Fujii, N.
Simultaneous stereoinversion and isomerization at the
Asp-4 residue in betaB2-crystallin from the aged human
eye lenses. Biochemistry, 50, 8628-35 (2011)[3] Carver, J. A., Cooper, P. G. and Truscott, R. J. (1993)
1H-NMR spectroscopy of beta B2-crystallin from bovine
eye lens. Conformation of the N- and C-terminal extensions. Eur. J. Biochem. 213, 313-320.

タンパク質中のアスパラギン酸残基の異性化と異常凝集
紀子、藤井 智彦、川口 剛、（金沢医大）佐々木 洋

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