粘虫谷氨酰胺转胺酶(MsTGase)活力测定条件的正交优化及其在幼虫体内的分布
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摘要
【目的】本研究旨在优化Grossowicz氧肟酸比色法测定粘虫Mythimna separata谷氨酰胺转胺酶(Ms TGase)活力的组合条件,以Ms TGase酶活力为依据分析其在不同龄期幼虫体内的分布规律。【方法】取4龄粘虫幼虫,通过组织匀浆和沉析纯化制备Ms TGase,采用Grossowicz比色法测定Ms TGase酶活力,并对Grossowicz比色法的多重实验因素进行正交优化,进一步结合差速离心法分析不同龄期幼虫体内和亚细胞组分(细胞核和细胞碎片,线粒体,微粒体以及胞质溶胶)中Ms TGase酶活力。【结果】结果表明,酶浓度、底物浓度、反应体系pH值、反应温度及钙离子浓度等实验因素都对Ms TGase酶活力测定结果产生显著影响,其影响大小顺序为:酶浓度>温度> p H>底物浓度>Ca2+浓度。Ms TGase酶比活力测定的最优化条件:酶浓度20 mg/m L、底物浓度0. 04 mol/L、反应体系pH值6. 5、测定温度37℃,不添加钙离子。在1-5龄幼虫中以4龄幼虫的Ms TGase酶活力最高,其比活力也显著高于其他龄期的,且在1-5龄幼虫胞质溶胶中Ms TGase酶活力分别占各亚细胞组分酶活力总和的39%,25%,48%,60%和61%。【结论】所获得的最优化条件适用于粘虫Ms TGase酶活力测定。Ms TGase在粘虫体内呈显著的龄期表达特征和亚细胞分布规律。
【Aim】This study aims to optimize the combination conditions in Grossowicz colorimetry for determining the activity of Mythimna separata transglutaminase(MsT Gase), and to analyze the distribution of MsT Gase in various larval instars based on its activity. 【Methods】MsT Gase was prepared from the 4 th instar larvae of M. separata,purified by tissue homogenate and precipitation,and assayed by Grossowicz colorimetry. The multiple experimental factors of Grossowicz colorimetric method were optimized by orthogonal experiment. The MsT Gase activities in larvae of different instars and subcellular fractions including cell nucleus and debris,mitochondria,microsomes and cytosol,which were separated by differential centrifugation,were further determined. 【Results 】 The experimental factors such as enzyme concentration,substrate concentration,pH value of the reaction system,temperature and calcium ion concentration had significant influences on the MsT Gase activity,and the effect of the experimental factors was ranked in the descending order as enzyme concentration > temperature > pH value > substrate concentration > Ca2 +concentration. The optimum conditions for MsT Gase activity determination included20 mg/mL of enzyme and 0. 04 mol/L of substrate in a reaction system of pH 6. 5,temperature 37℃,and no Ca2 +added. In the 1 st-5 th instar larvae of M. separata,the MsT Gase activity in the 4 th instar larvae was the highest and its specific activity was also significantly higher than those in other larval stages. The MsT Gase activities in the cytosol of the 1 st-5 th instar larvae of M. separata accounted for39%,25%,48%,60% and 61% of the total enzyme activity of each subcellular fraction,respectively.【Conclusion】The optimum conditions obtained are applicable for determining the MsT Gase activity.MsT Gase shows a significant instar-dependent accumulation and subcellular distribution in M. separata larvae.
【Aim】This study aims to optimize the combination conditions in Grossowicz colorimetry for determining the activity of Mythimna separata transglutaminase(MsT Gase), and to analyze the distribution of MsT Gase in various larval instars based on its activity. 【Methods】MsT Gase was prepared from the 4 th instar larvae of M. separata,purified by tissue homogenate and precipitation,and assayed by Grossowicz colorimetry. The multiple experimental factors of Grossowicz colorimetric method were optimized by orthogonal experiment. The MsT Gase activities in larvae of different instars and subcellular fractions including cell nucleus and debris,mitochondria,microsomes and cytosol,which were separated by differential centrifugation,were further determined. 【Results 】 The experimental factors such as enzyme concentration,substrate concentration,pH value of the reaction system,temperature and calcium ion concentration had significant influences on the MsT Gase activity,and the effect of the experimental factors was ranked in the descending order as enzyme concentration > temperature > pH value > substrate concentration > Ca2 +concentration. The optimum conditions for MsT Gase activity determination included20 mg/mL of enzyme and 0. 04 mol/L of substrate in a reaction system of pH 6. 5,temperature 37℃,and no Ca2 +added. In the 1 st-5 th instar larvae of M. separata,the MsT Gase activity in the 4 th instar larvae was the highest and its specific activity was also significantly higher than those in other larval stages. The MsT Gase activities in the cytosol of the 1 st-5 th instar larvae of M. separata accounted for39%,25%,48%,60% and 61% of the total enzyme activity of each subcellular fraction,respectively.【Conclusion】The optimum conditions obtained are applicable for determining the MsT Gase activity.MsT Gase shows a significant instar-dependent accumulation and subcellular distribution in M. separata larvae.
引文
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Bradford MM,1976.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal.Biochem.,72(1-2):248-254.
Eckert RL,Kaartinen MT,Nurminskaya M,Belkin AM,Colak G,Johnson GV,Mehta K,2014.Transglutaminase regulation of cell function.Physiol.Rev.,94(2):383-417.
Gao XW,Zhou XG,Wang RJ,Zheng BZ,1998.Distribution and purification of acetylcholinesterase in cotton bollworm(Lepidoptera:Noctuidae).Acta Entomol.Sin.,41(Suppl.):19-25.[高希武,周序国,王荣京,郑炳宗,1998.棉铃虫乙酰胆碱酯酶(AChE)的体躯分布及部分纯化.昆虫学报,41(增刊):19-25]
Grosswicz N,Wainfan E,Borek E,1950.The enzymatic formation of hydroxamic acids from glutamine and asparagines.J.Biol.Chem.,187(11):111-125.
Gundemir S,Colak G,Tucholski J,Johnson GV,2012.Transglutaminase 2:a molecular Swiss army knife.Biochim.Biophys.Acta Mol.Cell Res.,1823(2):406-419.
Kuo TF,Tatsukawa H,Kojima S,2011.New insights into the functions and localization of nuclear transglutaminase 2.FEBS J.,278(24):4756-4767.
Li P,Zhu YS,2002.Research progress of tissue transglutaminase.Foreign Med.Sci.:Mol.Biol.,24(5):294-297.[李平,朱运松,2002.组织型转谷氨酰胺酶的研究进展.国外医学(分子生物学分册),24(5):294-297]
Lindgren M,Riazi R,Lesch C,Wilhelmsson C,Theopold U,Dushay MS,2008.Fondue and transglutaminase in the Drosophila larval clot.J.Insect Physiol.,54(3):586-592.
Martin G,Rita C,Bergamini CM,2003.Transglutaminases:nature’s biological glues.Biochem.J.,368(Pt 2):377-396.
Matsumura Y,Chanyongvorakul Y,Kumazawa Y,Ohtsuka T,Mori T,1996.Enhanced susceptibility to tranglutaminase reaction ofα-lactalbumin in the molten globule state.Biochim.Biophys.Acta,1292(1):69-76.
Nsango SE,Pompon J,Xie T,Rademacher A,Fraiture M,Thoma M,Awono-Ambene PH,Moyou RS,Morlais I,Levashina EA,2013.AP-1/Fos-TGase2 axis mediates wounding-induced Plasmodium falciparum killing in Anopheles gambiae.J.Biol.Chem.,288(22):16145-16154.
Nurminskaya MV,Belkin AM,2012.Cellular functions of tissue transglutaminase.Int.Rev.Cell Mol.Biol.,294:1-97.
Phillips DR,Clark KD,2017.Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition,melanization,coagulants,and hemocyte recruitment.PLoS ONE,12(2):e0171447.
Shibata T,Kawabata SI,2018.Pluripotency and a secretion mechanism of Drosophila transglutaminase.J.Biochem.,163(3):165-176.
Silveira H,Gabriel A,Ramos S,Palma J,Felix R,Custódio A,Collins LV,2012.CpG-containing oligodeoxynucleotides increases resistance of Anopheles mosquitoes to Plasmodium infection.Insect Biochem.Molec.Biol.,42(10):758-765.
Singh RN,Mehta K,1994.Purification and characterization of a novel transglutaminase from filarial nematode Brugia malayi.Eur.J.Biochem.,225(2):625-634.
Tsai YH,Lai WF,Chen SH,Johnson LR,1998.A novel calciumindependent enzyme capable of incorporating putrescine into proteins.Biochem.Biophy.Res.Commun.,244(1):161-166.
Wang SP,Liu JH,2010.Research progress of transglutaminase.Hunan Agric.Sci.,(15):41-43.[王书平,刘俊华,2010.谷氨酰胺转胺酶的研究进展.湖南农业科学,(15):41-43]
Zhang L,Rao WB,Muhayimana S,Zhang XF,Xu JY,Xiao CY,Huang QC,2018.Purification and biochemical characterization of a novel transglutaminase from Mythimna separata larvae(Noctuidae,Lepidoptera).J.Biotechnol.,265:1-7.
Beninati S,Bergamini CM,Piacentini M,2009.An overview of the first50 years of transglutaminase research.Amino Acids,36(4):591-598.
Bradford MM,1976.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal.Biochem.,72(1-2):248-254.
Eckert RL,Kaartinen MT,Nurminskaya M,Belkin AM,Colak G,Johnson GV,Mehta K,2014.Transglutaminase regulation of cell function.Physiol.Rev.,94(2):383-417.
Gao XW,Zhou XG,Wang RJ,Zheng BZ,1998.Distribution and purification of acetylcholinesterase in cotton bollworm(Lepidoptera:Noctuidae).Acta Entomol.Sin.,41(Suppl.):19-25.[高希武,周序国,王荣京,郑炳宗,1998.棉铃虫乙酰胆碱酯酶(AChE)的体躯分布及部分纯化.昆虫学报,41(增刊):19-25]
Grosswicz N,Wainfan E,Borek E,1950.The enzymatic formation of hydroxamic acids from glutamine and asparagines.J.Biol.Chem.,187(11):111-125.
Gundemir S,Colak G,Tucholski J,Johnson GV,2012.Transglutaminase 2:a molecular Swiss army knife.Biochim.Biophys.Acta Mol.Cell Res.,1823(2):406-419.
Kuo TF,Tatsukawa H,Kojima S,2011.New insights into the functions and localization of nuclear transglutaminase 2.FEBS J.,278(24):4756-4767.
Li P,Zhu YS,2002.Research progress of tissue transglutaminase.Foreign Med.Sci.:Mol.Biol.,24(5):294-297.[李平,朱运松,2002.组织型转谷氨酰胺酶的研究进展.国外医学(分子生物学分册),24(5):294-297]
Lindgren M,Riazi R,Lesch C,Wilhelmsson C,Theopold U,Dushay MS,2008.Fondue and transglutaminase in the Drosophila larval clot.J.Insect Physiol.,54(3):586-592.
Martin G,Rita C,Bergamini CM,2003.Transglutaminases:nature’s biological glues.Biochem.J.,368(Pt 2):377-396.
Matsumura Y,Chanyongvorakul Y,Kumazawa Y,Ohtsuka T,Mori T,1996.Enhanced susceptibility to tranglutaminase reaction ofα-lactalbumin in the molten globule state.Biochim.Biophys.Acta,1292(1):69-76.
Nsango SE,Pompon J,Xie T,Rademacher A,Fraiture M,Thoma M,Awono-Ambene PH,Moyou RS,Morlais I,Levashina EA,2013.AP-1/Fos-TGase2 axis mediates wounding-induced Plasmodium falciparum killing in Anopheles gambiae.J.Biol.Chem.,288(22):16145-16154.
Nurminskaya MV,Belkin AM,2012.Cellular functions of tissue transglutaminase.Int.Rev.Cell Mol.Biol.,294:1-97.
Phillips DR,Clark KD,2017.Bombyx mori and Aedes aegypti form multi-functional immune complexes that integrate pattern recognition,melanization,coagulants,and hemocyte recruitment.PLoS ONE,12(2):e0171447.
Shibata T,Kawabata SI,2018.Pluripotency and a secretion mechanism of Drosophila transglutaminase.J.Biochem.,163(3):165-176.
Silveira H,Gabriel A,Ramos S,Palma J,Felix R,Custódio A,Collins LV,2012.CpG-containing oligodeoxynucleotides increases resistance of Anopheles mosquitoes to Plasmodium infection.Insect Biochem.Molec.Biol.,42(10):758-765.
Singh RN,Mehta K,1994.Purification and characterization of a novel transglutaminase from filarial nematode Brugia malayi.Eur.J.Biochem.,225(2):625-634.
Tsai YH,Lai WF,Chen SH,Johnson LR,1998.A novel calciumindependent enzyme capable of incorporating putrescine into proteins.Biochem.Biophy.Res.Commun.,244(1):161-166.
Wang SP,Liu JH,2010.Research progress of transglutaminase.Hunan Agric.Sci.,(15):41-43.[王书平,刘俊华,2010.谷氨酰胺转胺酶的研究进展.湖南农业科学,(15):41-43]
Zhang L,Rao WB,Muhayimana S,Zhang XF,Xu JY,Xiao CY,Huang QC,2018.Purification and biochemical characterization of a novel transglutaminase from Mythimna separata larvae(Noctuidae,Lepidoptera).J.Biotechnol.,265:1-7.