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1 PERIODICAL OF OCEAN UNIVERSITY OF CHINA 37 (4) : J uly, 2007 Ξ, ΞΞ, (, ) :, ( A postichopus japonicus) (LDH) ( EST) (SOD) LDH 10 20,12 h EST SOD, h EST 3 h SOD,EST SOD 1,, : ; ; ; : S : A : (2007) , [1 ] [225 ] (LDH), [5 ], [6 ],LDH, LDH [7 ] ( EST) [8 ] (SOD), [9210 ],SOD, SOD [11 ] ( A postichopus japonicus), [12213 ],, [14215 ],,,, 1 ( 2. 5 g) , ; , h,1 h,3 h,12 h,24 h,72 h 10, - 80, , (18 2), (18 4), (18 6) ( 1), d, 10, Fig. 1 Diagram of the diel temperature fluctuating mode Ξ ΞΞ : ( ) : ; : : (19812),,, E2mail :pinkhum - com : E2mail : edu. cn

4, : 587 1. 3,, ( 0. 05 mol/ L,pH = 75) 4 3 000 g 5 min, -, - 20 1. 4 3 10 % 4. 5 % Tris2HCl (0. 5 mol/ L,p H = 6. 8) Tris2HCl (1. 5 mol/ L, p H = 8. 8) Tris2 (p H = 8.

0), 37,, 7 % [18 ] : 2. 45 10-3 mol/ L (NB T) 20 min, 0. 028 mol/ L ( TEMED) 2. 8 10-5 mol/ L 0. 030 mol/ L (p H = 7. 8) 15 min, 0. 05 mol/ L (ph = 7.

2 Electrophoretograms of EST isozyme at acute temperature changes, (showed the deep color band, Level 1 band) ;, (showed the light color band, Level 2 band) ;, ; (showed the lightest color band,

2 4, : ,, ( mol/ L,pH = 75) g 5 min, -, % 4. 5 % Tris2HCl (0. 5 mol/ L,p H = 6. 8) Tris2HCl (1. 5 mol/ L, p H = 8. 8) Tris2 (p H = 8. 3) 4, 20 ma, 40 ma, 1 cm [16 ] : NAD 40 mg, ml,pms 2 mg,nb T 20 mg,0. 5 mol/ L (p H = 7. 5) 10 ml,0. 1 mol/ L NaCl 5 ml, 50 ml [17 ] : 50 mg 2 50 mg 2, 50 mg,, 50 ml 0. 1 mol/ L (ph = 7. 0), 37,, 7 % [18 ] : mol/ L (NB T) 20 min, mol/ L ( TEMED) mol/ L mol/ L (p H = 7. 8) 15 min, mol/ L (ph = 7. 8) 10-4 mol/ L EDTA, lx 30 min, [17 ], 1, EST1, 20 10,1 h EST2, 3 h EST2, ( 2) 2 3 Fig. 2 Electrophoretograms of EST isozyme at acute temperature changes, (showed the deep color band, Level 1 band) ;, (showed the light color band, Level 2 band) ;, ; (showed the lightest color band, Level 3 band ; the band at every levels di2 vided into the brand band and the narrow band, according to the band width) SOD S21, 10 20, 1 h S22 S23,12 h S22 24 h h 3 h S22 S23 12 h ( 3) 3 SOD 3 Fig. 3 Electrophoretograms of SOD isozyme at acute temperature changes 2 2, LDH, EST SOD 2. 1 EST SOD EST EST ,EST2,12 h EST2, 24 h EST2, 4 33 Fig. 4 Electrophoretograms of EST isozyme at different amplitudes of temperature change h,1 h,3 h,12 h,24 h,72 h h 1 h 3 h 12 h 24 h 72 h Lanes 1 6, electrophoretograms at 0 h, 1 h, 3 h, 12 h, 24 h and 72 h heat exposure (from 10 to 20 ), respectively ; Lanes 7 12, electrophoretograms at 0 h, 1 h, 3 h, 12 h, 24 h and 72 h ( Fig. 2 Fig. 5) 1 2 (18 0) 3 4 (18 2) 5 6 (18 4) 7 8 (18 6)

588 2 0 0 7 2. 2 EST SOD, EST SOD 1, ( 4, 5) 5 33 Fig.

3 EST SOD, EST SOD 1, ( 4, 5) 5 33 Fig. 5 Electrophoretograms of SOD isozyme at different 3 amplitudes of temperature change,, [19220 ], [21 ], [22 ], [20 ],, [23 ],,, 1 [24 ], h,, 12 h h,,,,,est2 12 h,,, EST2 1 h,, [14 ] [12 ],, [12 ], (reactive oxygen species, ROS) [25226 ],, SOD,SOD, [27 ] [28 ] [29 ] SOD,, [30 ], SOD [31 ], (ROS) [32 ] ROS [33 ], SOD SOD,,,, ROS, [34 ], ROS, [26 ] [35 ], ( SOD) [36 ], EST SOD 3, 70 d, 18,(18 2),, [15 ] 45 d,2, EST SOD,,,EST SOD,, 25 g [37 ], [12 ],LDH,,, LDH [38 ]

4 4, : 589 LDH,, SOD EST,,,,,,, : [ 1 ] Zheng X D, Zhao J M, Xiao S, et al. Isozymes analysis of the golden Cut2 tlefish Sepia esculenta (Cephalopoda : Sepiidae) [J ]. J Ocean Univ. China (in English), 2004, 3 (1) : [2 ],,,. [J ]. :, 2003, 33 (1) : [3 ],,. [J ] , 24 (1) : [4 ],. [J ]., 1999, 6 (1) : [5 ],. 6 [J ]., 1997, 19 (2) : [6 ],,. Paecilomyces inflatus LDH [J ]. ( ), 1998, 31 (2) : [7 ] Yamawaki H, Tsukuda H. Significance of the variation in isozymes of liver lactate dehydrogenase with thermal acclimation in goldfish I. thermosta2 bility and temperature dependency [J ]. Comp Biochem Physiol, 1979, 62B (1) : [8 ],,,. 3 [J ]., 2005, 20 (2) : [9 ],. [J ]., 1999, 6 (1) : [10 ] Lenartova V, Holouska K, Pedrajas J R, et al. Antioxidant and detoxify2 ing fish enzymes as biomarkers of river pollution [J ]. Biomarkers, 1997, 2 (4) : [11 ] Pedrajas J R, Gavilanes F, L pez2barea J, et al. Incubation of superoxide dismutase with malondialdehyde and 42hydroxy222nonenal forms new ac2 tive isoforms and adducts An evaluation of xenobiotics in fish [J ]. Chem Biol Interact, 1998, 116 : [12 ],. [J ]., 2004, 19 (1) : [13 ] Kanno M, Kijima A. Genetic differentiation among three color variants of Japanese sea cucumber Stichopus japonicas [J ]. Fish Sci, 2003, 69 (4) : [14 ],,,. [J ]., 2005, 12 (1) : [15 ],,,. [J ]., 2005, 29 (5) : [16 ] Zietara M S, Skorkowski E F. Unusual expression of the threespine stickleback ( Gasterosteus aculeatus) lactate dehydrogenase isoenzymes and partial characterization of purified LDH2A4 [J ]. Comp Biochem Physiol, 1991, 99B : [17 ],. [ M]. :, [18 ],. [J ]., 2003, 12 (6) : [19 ],,. [J ]., 1998, 28 (3) : [20 ] Hochachka P W, Somero G N. Biochemical Adaptation : Mechanism and Process in Physiological Evolution [ M ]. Press, Oxford : Oxford University [21 ] Rogers K D, Seebacher F, Thompson M B. Biochemical acclimation of metabolic enzymes in response to lowered temperature in tadpoles of Limnodynastes peronii [J ]. Comp Biochem Physiol, 2004, 137A : [22 ],,. (ABA) [J ]., 1999, 13 (4) : [ 23 ] Rau M A, Whitaker J, Freedman J H, et al. Differential susceptibility of fish and rat liver cells to oxidative stress and cytotoxicity upon exposure to prooxidants [J ]. Comp Biochem Physiol, 2004, 137C: [24 ] Lushchak VI, Bagnyukova TV. Temperature increase results in oxidative stress in goldfish tissue. 1. indices of oxidative stress [J ]. Comp Biochem Physiol, 2006, 143C: [25 ] Lemaire P, Livingstone D R. Pro2oxidant/ antioxidant processes and or2 ganic xenobiotic interactions in marine organisms, in particular the floun2 der Platichthys f lesus and the mussel Mytilus edulis [J ]. Trends Comp Biochem Physiol, 1993, 1 : [26 ] Winston G W, Di Giulio R T. Prooxidant and antioxidant mechanisms in aquatic organism [J ]. Aquat Toxicol, 1991, 19 : [27 ],,. SOD [J ]., 2004, 40 (6) : [28 ] Parihar M S, Javeri T, Hemnani T, et al. Responses of superoxide dis2 mutase, glutathione antioxidant defenses in gills of the freshwater catfish ( Heteropneustes Fossilis ) to short2term elevated temperature [ J ]. J therm Biol, 1997, 22 (2) : [29 ] Zhang L, Onda K, Imai R, et al. Growth temperature downshift induces antioxidant response in Saccharomyces cerevisiae [J ]. Biochem Biophys Res Commun, 2003, 307 (2) : [30 ] Thieringer H A, Jones P G, Inouye M. Cold shock and adaptation [J ]. Bio Essays, 1998, 20 : [31 ],,,. [J ]., 2002, 33 (2) : [32 ] Boveris A, Cadenas E, Stoppani A O. Role of ubiquinone in the mito2 chondrial generation of hydrogen peroxide [J ]. J Biochem, 1976, 156 : [33 ] Sies H. Oxidative stress : introduction [ A ]. In : Sies H. Oxidative Stress: Oxidants and Antioxidants [ C ]. San Diego : Academic Press, 1991, [34 ] Parihar M S, Dubey A K. Lipid peroxidation and ascorbic acid status in respiratory organs of male and female freshwater catfish Heteropneustes fossilis exposed to temperature increase [J ]. Comp Biochem Physiol,

5 , 112C: [35 ] Gutteridge J M, Halliwell B. The measurement and mechanism of lipid peroxidation in biological systems [ J ]. Trends Biochem Sci, 1990, 15 (4) : [36 ] Parihar M S, Dubey A K, Faveri T, et al. Changes in lipid peroxidation, superoxide dismutase activity, ascorbic acid and phospholipids content in liver of freshwater catfish Heteropneustes Fossilis exposed to elevated tem2 perature [J ]. J therm Biol, 1996, 21 : [37 ],,,. ( A postichopus japonicus Selenka) 1 [J ]., 1996, 3 (2) : [38 ]. [D]. :, Effects of Temperature Changes on Isozymes in the Sea Cucumber, Apostichopus japonicus J I Ting2Ting, DON G Yun2Wei, DON G Shuang2Lin ( Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao , China) Abstract : The effects of acute temperature change and diel fluctuating temperature on the isozyme patterns of LDH, EST and SOD of t he sea cucumber A postichopus japonicus were st udied. temperature change, young A. In t he experiment of acute japonicus were subjected to heat exposure (from 10 to 20 ) and cold expo2 sure (from 20 to 10 ), respectively. The samples were taken at 0 h, 1 h, 3 h, 12 h, 24 h and 72 h after heat or cold exposure, respectively. In t he experiment of diel fluct uating temperat ures, young A. japonicus were subjected to four temperature treatments ( 18, 18 2, 18 4 and 18 6 ). The results showed that there was no expression of LDH isozyme. In the experiment of acute temperature changes, within 12 h heat exposure, there were new isomers that appeared both in EST and SOD. After 1 h cold exposure, there was one new band EST2 in the EST isozyme pattern. After 3 h cold exposure there were two new bands band S2 and band S3 in the SOD isozyme pattern. were no changes in t he patterns of EST and SOD isozymes. In the experiment of diel fluctuating temperature, there Key words : A postichopus japonicus ; temperature change ; isozyme ; LDH ; EST ; SOD

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