Digestion of thioredoxin and 1
5 L.-E. Shao, M. B. Diccianni, T. Tanaka, R. Gribi, A. L. Yu, J. D.
Pullen, B. M. Camitta and J. Yu, Cancer Res., 2001, 61, 7333.
To 40 ll of diluted thioredoxin and a solution of 1 (concen-
6
Y. Soini, K. Kahlos, U. Napankangas, R. Kaarteenaho-Wiik, M.
Saily, P. Koistinen, P. Paakko, A. Holmgren and V. L. Kinnula, Clin.
Cancer Res., 2001, 7, 1750.
−
1
tration: 40 pmol ll ) was added urea (6 M) and the mixture
◦
was heated at 95 C for 20 min. Then we added 150 ll of
7
8
9
J. Raffel, A. K. Bhattacharyya, A. Gallegos, H. Cui, J. G. Einspahr,
D. S. Alberts and G. Powis, J. Lab. Clin. Med., 2003, 142, 46.
D. T. Lincoln, E. M. Ali Emadi, K. F. Tonissen and F. M. Clarke,
Anticancer Res., 2003, 23, 2425.
J. Wang, M. Kobayashi, K. Sakurada, M. Imamura, T. Moriuchi and
M. Hosokawa, Blood, 1997, 89, 2480.
5
0 mM NH
4
HCO
3
buffer and 1.0 ll of the chymotrypsin solution
◦
(
containing 2 lg of enzyme) and incubated at 37 C overnight.
After digestion, the peptides were purified with a ZipTip C18
pipette tip.
GHOST (Glutathione as substrate of thioredoxin) assay
10 N. Kawahara, T. Tanaka, A. Yokomizo, H. Nanri, M. Ono, M. Wada,
K. Kohno, K. Takenaka, K. Sugimachi and M. Kuwano, Cancer
Res., 1996, 56, 5330.
11 A. Yokomizo, M. Ono, H. Nanri, Y. Makino, T. Ohga, M. Wada, T.
Okamoto, J. Yodoi, M. Kuwano and K. Kohno, Cancer Res., 1995,
In a total assay volume of 3 ml, 10 ll of NADPH (to 100 lM)
was mixed with buffer, thioredoxin (to 5.0 lM) and E. coli
thioredoxin reductase (10 nM). The enzymatic reaction was
initiated by adding 150 ll of GSSG (to 1 mM).
5
5, 4293.
1
1
1
2 T. Sasada, S. Iwata, N. Sato, Y. Kitaoka, K. Hirota, K. Nakamura,
A. Nishiyama, Y. Taniguchi, A. Takabayashi and J. Yodoi, J. Clin.
Invest., 1996, 97, 2268.
Physical measurements
3 D. L. Kirkpatrick, M. Kuperus, M. Dowdeswell, N. Potier, L. J.
Donald, M. Kunkel, M. Berggren, M. Angulo and G. Powis,
Biochem. Pharmacol., 1998, 55, 987.
4 P. Wipf, S. M. Lynch, A. Birmingham, G. Tamayo, A. Jimenez, N.
Campos and G. Powis, Org. Biomol. Chem., 2004, 2, 1651.
15 M. Pallis, T. D. Bradshaw, A. D. Westwell, M. Grundy, M. F. G.
Matrix-assisted laser desorption-ionization time of flight mass
spectrometry (MALDI-TOF MS) spectra were recorded using a
Bruker ultraflex TOF/TOF. MALDI spots were made by mixing
−
1
−1
5
ll of 20 pmol ll of complex solution or 5 pmol ml of diges-
tion solution with 5 ll of matrix solution. 1 ll of this solution
was deposited on a stainless steel probe and allowed to air-dry.
The matrix solution was 0.07% trifluoroacetic acid and 30%
acetonitrile solution saturated a-cyano-4-hydroxycinnamic acid
Stevens and N. Russell, Biochem. Pharmacol., 2003, 66, 1695.
1
1
6 S. Gromer, S. Urig and K. Becker, Med. Res. Rev., 2003, 24, 40.
7 F. Rollin-Genetet, C. Berthomieu, A.-H. Davin and E. Quemeneur,
Eur. J. Biochem., 2004, 271, 1299.
(
CHCA) or sinapic acid (SA). UV-visible spectra were recorded
1
8 S. Gromer, L. D. Arscott, C. H. Williams, Jr., R. H. Schirmer and K.
Becker, J. Biol. Chem., 1998, 273, 20096.
19 K. Becker, C. Herold-Mende, J. J. Park, G. Lowe and R. H. Schirmer,
on a Shimadzu UV3100PC spectrometer. These measurements
were carried out using cells of 0.1- and 1.0-cm pathlength.
Circular dichroism (CD) measurements were recorded on a
Jasco J-720 w spectropolarimeter. Measurement was carried out
using a 0.01 cm pathlength cell at ca. 303 K.
J. Med. Chem., 2001, 44, 2784.
0 S. Bonse, J. M. Richards, S. A. Ross, G. Lowe and R. L. Krauth-
Siegel, J. Med. Chem., 2000, 43, 4812.
1 J. A. Weinstein, N. N. Zheligovskaya, M. Y. Mel’nikov and F. Hartl,
J. Chem. Soc., Dalton Trans., 1998, 2459.
2
2
Acknowledgements
22 J. A. Weinstein, A. J. Blake, E. S. Davies, A. L. Davis, M. W. George,
D. C. Grills, I. V. Lileev, A. M. Maksimov, P. Matousek, M. Y.
Mel’nikov, A. W. Parker, V. E. Platonov, M. Towrie, C. Wilson and
N. N. Zheligovskaya, Inorg. Chem., 2003, 42, 7077.
This work was supported by a Grant-in Aid for Scientific
Research on Priority Area (A) (No. 12304040) from the Ministry
of Education, Culture, Sports, Science and Technology, Japan.
One of the authors (M. K.) expresses his special thanks for the
center of excellence (21COE) program “Creation of Integrated
EcoChemistry of Osaka University”.
23 S. K. Katti, D. M. LeMaster and H. Eklund, J. Mol. Biol., 1990, 212,
1
67.
2
2
2
4 A. Weichsel, J. R. Gasdaska, G. Powis and W. R. Montfort, Structure,
1
5 R. Friemann, H. Schmidt, S. Ramaswamy, M. Forstner, R. L.
Krauth-Siegel and H. Eklund, FEBS Lett., 2003, 554, 301.
6 H. J. Dyson, G. P. Gippert, D. A. Case, A. Holmgren and P. E.
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1
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