Science of Sericulture

Science of Sericulture 2012 38 1 0065-0069 ISSN 0257-4799 CN 32-1115 /S E-mail CYKE@ chinajournal. net. cn 215123 25 0 ~ 24 h GSH GSSG GSH + 2GSSG GSH /GSSG S- GST TPX 23% GR 57% GSH TPX GST GSSG 61% GSH + 2GSSG 41% GSH /GSSG 33% GR 16% GSH GSSG GSH + 2GSSG GSH /GSSG TPX GST GR GPX TrxR TPX GR GSH /GSSG S881. 2 A 0257-4799 2012 01-0065 - 05 An Analysis on Status of Glutathione Redox Cycle Between Diapause and Nondiapause Eggs of the Silkworm Bombyx mori Before Diapause Initiation WAN Hua-Xing SIMA Yang-Hu ZHAO Lin-Chuan * School of Biology and Basic Medicine Sciences Soochow University Suzhou Jiangsu 215123 China Abstract In order to understand whether the glutathione redox cycle is different in diapause eggs and non-diapause eggs of the silkworm Bombyx mori before glutathione content and activity of related metabolic enzymes in diapause and non-diapause eggs before from 0 h to 24 h post-oviposition at 25 were determined using spectrophotometric methods. It was discovered that before diapausing eggs had no significant variation in contents of reduced glutathione GSH oxidized glutathione GSSG and total glutathione GSH +2GSSG in ratio of GSH /GSSG and in activity of glutathione S-transferase GST. However the activity of thioredoxin peroxidase TPX was reduced by 23% and the activity of glutathione reductase GR was increased by 57%. Non-diapausing eggs had no significant variation in GSH content and activity of TPX and GST either. But GSSG content was increased by 61% GSH +2GSSG content was increased by 41% the ratio of GSH /GSSG was reduced by 33% and activity of GR was reduced by 16%. Compared to non-diapausing eggs diapausing eggs had lower contents of GSH GSSG and GSH + 2GSSG higher ratio of GSH / 2011-10 - 23 2011-11 - 13 No. 30571406 1986 - E-mail wanhuaxingsd@ 163. com E-mail sdzlc2008@ 126. com * Corresponding author. E-mail sdzlc2008@ 126. com GSSG lower activity of TPX no significant difference in activity of GST and higher activity of GR. However activity of glutathione peroxidase GPX and thioredoxin reductase TrxR was undetectable in both diapause and non-diapause eggs. These results indicate that higher ratio of GSH /GSSG in diapause eggs before diapause initi-

66 2012 38 1 ation is resulted from lower activity of TPX and higher activity of GR making the glutathione redox cycle remain at a relatively reduced state. Key words Bombyx mori egg Diapause initiation Glutathione redox cycle Thioredoxin peroxidase Glutathione reductase GSH GSSG 1-2 GPX TPX GST GR TrxR glutathione peroxidase GPX 1 thioredoxin peroxidase TPX S- glutathione S-transferase GST 1. 1 reduced glutathione GSH H 2 O 2 25 GSH oxidized 15 2 glutathione GSSG 25 GSH 12L 12D glutathione reductase GR 25 12L 12D thioredoxin reductase TrxR 0 6 12 18 24 h reducing - 80 nicotinamide adenine dinucleotide phosphate NADPH H 2 O 2 CAT mrna 3 GSSG GSH 3-4 GSH / GSSG 1. 2 5 1. 2. 1 0. 1 g 25 24 h 4 0. 05 mol /L PBS ph 7. 0 5% 1. 0 ml 4 10 000 g 8 min 6 - G 2 7 GSH GSSG H 2 O 2 H 2 O 2 1. 2. 2 0. 1 g catalase CAT mrna 4 0. 05 mol /L PBS ph 7. 0 5% GSH /GSSG TPX 1. 0 ml 4 10 000 g 8 min 4 10 000 g 4 min GPX GSH /GSSG TPX GR TrxR 4 8 - TPX 14 GPX TPX GR TrxR 25 1 min 1 μmol NADPH mg GST 10 G-250 1. 3 ANOVA Student's Newman-Keuls post hoc tests P < 0. 05

1 67 P < 0. 01 2 2. 1 2. 1. 1 1 0 ~ 24 h GSH GSH P < 0. 05 ** P < 0. 01 3 ~ 5 7 ** means extremely significant difference P < 0. 01. 2 Fig. 2 The same in Fig. 3 ~ 5 7. Variations of GSSG content between diapausing Student's test * P < 0. 05 4 Significance of difference in biochemical indexes at the same time between diapausing and non-diapausing eggs was determined by Student's test. 1 Fig. 1 * means significant difference P < 0. 05. The same in Fig. 4. Variations of GSH content between diapausing 2. 1. 3 3 0 ~ 24 h 41% P < 0. 01 2. 1. 2 2 Fig. 3 Variations of total glutathione between diapausing 0 ~ 24 h GSSG GSSG 61% GSSG P < 0. 01 2. 1. 4 4 0 ~ 24 h 3 GSH /GSSG GSH /GSSG 33% GSH /GSSG

68 2012 38 1 4 6 S- Fig. 4 Variations of GSH / GSSG ratio between Fig. 6 Variations of GST activity between diapausing diapausing and non-diapausing silkworm eggs before 2. 2 2. 2. 3 7 0 ~ 24 h GR 57% 2. 2. 1 GR 16% 0 ~ 24 h GR P < 0. 01 GPX TPX 5 0 ~ 24 h TPX 23% TPX TPX P < 0. 01 7 Fig. 7 Variations of GR activity between diapausing 5 3 Fig. 5 Variations of TPX activity between diapause and non-diapause silkworm eggs before 2 mol GSH 1 mol GSSG GSH + 2GSSG γ- 2. 2. 2 S- 6 glutamate cysteine ligase GCL 0 ~ 24 h GST glutathione synthase GS GSH GST glutamyltransferase GT GSH GST γ-

1 69 GST GSH TPX GR GSH GSSG 2 25 0 ~ 24 h diapause hormone GST DH γ- 6 References 41% GST γ- GSH 1 Forman H J Zhang H Rinna A. Glutathione overview of its protective roles measurement and biosynthesis J. Mol Aspects Med 2009 30 1 /2 1-12 GST 2 Lu S C. Regulation of glutathione synthesis J. Mol Aspects Med 2009 30 1 /2 42-59 γ- GSH GSSG J. Science 4 Krishnan N GSH GSSG 61% γ- Biol 2009 GSH GSH 5 Asensi M GSSG GSH GSSG GSH GSSG GSH /GSSG structure 5 7 GPX TPX GST GSH GSSG GR TrxR GSSG GSH 3-4 9 GPX TrxR GPX TrxR 2010 36 4 697-701 10 GST J. 2010 36 1 11 GST J. 2010 53 12 TPX et al. Variation in glutathione status associated with induction and initiation of diapause in eggs of the bi- GR TPX J. 2011 37 1 GSH GSSG 13 Meng G Yao J M Wang L GR voltine strain of the silkworm Bombyx mori J GSSG GSH 2011 36 2 173-179 14 Sima Y H Yao J M Hou Y H et al. Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initia- tion and termination J 2 72-80 3 Kanzok S M Fechner A Bauer H et al. Substitution of the thioredoxin system for glutathione reductase in Drosophila melanogaster 2001 291 5504 643-646 Kodric D Kiudkiewicz B et al. Glutathione-ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata Say J. Insect Biochem Mol 39 3 180-188 Sastre J Pallardo F V et al. Ratio of reduced to oxidized glutathione as indicator of oxidative stress and DNA damage J. Methods Enzymol 1999 299 267-276 6 Yamashita O. Diapause hormone of the silkworm Bombyx mori 42 7 669-679 1 1-6 gene expression and function J. J Insect Physiol 1996. J. 2003 29 8. J. 2009 52 6 604-609. H 2 O 2 J.. S- 170-174. 1333-1338 12. 52-56. Physiol Entomol. Arch Insect Biochem Physiol 2011 77