圆二色谱法分析金属离子对鲍鱼脯氨酰内肽酶的作用
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摘要
使用原核表达系统表达了皱纹盘鲍(Haliotis discus hannai)脯氨酰内肽酶(prolyl endopeptidase,PEP),并对其进行高度纯化。SDS-PAGE结果表明,该酶的相对分子质量约为85 ku,其二级结构主要由α-螺旋(28.3%)、反向平行结构(17.4%)、平行结构(8.70%)、β转角(19.0%)、无规卷曲(28.5%)组成。选取PEP特异性荧光底物Suc-Gly-Pro-MCA测定其活性,并选取了多种常见金属盐溶液对其进行抑制作用分析。结果发现,Al~(3+)、Cu~(2+)、 Zn~(2+)、Ag~+和Pb~(2+)能够抑制PEP的活性。利用圆二色谱法,对金属离子作用下PEP的二级结构变化进行分析和计算。分析结果表明,不同金属离子对PEP的作用效果不同:Al~(3+)、Cu~(2+)和Zn~(2+)在影响PEP结构时也影响其活性;Ca~(2+)、Mg~(2+)对PEP的结构和活性均不产生明显影响,而Ag~+和Pb~(2+)可在不影响PEP二级结构的前提下抑制其活性。
Purified recombinant PEP from abalone(Haliotis discus hannai) was obtained using a prokaryotic expression system,with a relative molecular mass of 85 ku.The secondary structure of PEP mainly contained of alpha-helix-28.3%,antiparallel-17.4%,parallel-8.70%,beta-turn-19.0% and random coil-28.5%.The measurement of enzyme activity was based on a specific fluorogenic substrate Suc-Gly-Pro-MCA and a variety of common metal salts solution was selected for the inhibition assay.The results showed that the enzyme activity of PEP was strongly inhibited by Al~(3+),Cu~(2+),Zn~(2+),Ag~+ and Pb~(2+).The secondary structure of PEP treated with metal ions was analyzed using circular dichroism spectrum.The influence of various metal ions on the PEP activity was different.In comparison,Al~(3+),Cu~(2+) and Zn~(2+) had an impact on both enzyme activity and the secondary structure,while Ca~(2+) and Mg~(2+) did not influence the secondary structure and activity obviously.Ag~+ as well as Pb~(2+) revealed a strong inhibition on enzyme activity while less influence on the secondary structure.
Purified recombinant PEP from abalone(Haliotis discus hannai) was obtained using a prokaryotic expression system,with a relative molecular mass of 85 ku.The secondary structure of PEP mainly contained of alpha-helix-28.3%,antiparallel-17.4%,parallel-8.70%,beta-turn-19.0% and random coil-28.5%.The measurement of enzyme activity was based on a specific fluorogenic substrate Suc-Gly-Pro-MCA and a variety of common metal salts solution was selected for the inhibition assay.The results showed that the enzyme activity of PEP was strongly inhibited by Al~(3+),Cu~(2+),Zn~(2+),Ag~+ and Pb~(2+).The secondary structure of PEP treated with metal ions was analyzed using circular dichroism spectrum.The influence of various metal ions on the PEP activity was different.In comparison,Al~(3+),Cu~(2+) and Zn~(2+) had an impact on both enzyme activity and the secondary structure,while Ca~(2+) and Mg~(2+) did not influence the secondary structure and activity obviously.Ag~+ as well as Pb~(2+) revealed a strong inhibition on enzyme activity while less influence on the secondary structure.
引文
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[19]卢斌,柯才焕,王文雄.低浓度镉、锌暴露对白氏文昌鱼的毒性累积及其几种重要酶活性的影响[J].厦门大学学报(自然科学版),2012,51(4):767-773.
[2]MORIYAMA A,NAKANISHI M,SASAKI M.Porcine muscle prolyl endopeptidase and its endogenous substrates[J].Journal of Biochemistry,1988,104:112-118.DOI:10.1093/oxfordjournals.jbchem.a122404.
[3]YOSHIMOTO T,NISHIMURA T,KITA T,et al.Post-proline cleaving enzyme(prolyl endopeptidase) from bovine brain[J].Journal of Biochemistry,1983,94:1179-1190.DOI:10.1093/oxfordjournals.jbchem.a134463.
[4]GASS J,KHOSLA C.Prolyl endopeptidases[J].Cellular and Molecular Life Sciences,2007,64:345-355.
[5]FULOP V,BOCSKEI Z,POLGAR L.Prolyl oligopeptidase:an unusual beta-propeller domain regulates proteolysis[J].Cell,1998,94(2):161-170.DOI:10.1016/S0092-8674(00)81416-6.
[6]KAROL K,TOMASZ R,REINIS D.Molecular dynamics,crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase[J].Biochimie,2012,94(6):1398-1411.DOI:10.1016/j.biochi.2012.03.012.
[7]GORKA L,ITXARO P,LORENA B.Prolyl endopeptidase activity is correlated with colorectal cancer prognosis[J].International Journal of Medical Sciences,2014,11(2):199-208.DOI:10.7150/ijms.7178.
[8]YOSHIDA K,INABA K,OHTAKE H.Purification and characterization of prolyl endopeptidase from the Pacific herring,Clupea pallasi,and its role in the activation of sperm motility[J].Development Growth & Differentiation,1999,41(2):217-225.DOI:10.1046/j.1440-169x.1999.00424.x.
[9]WANG M X,ZHONG C,CAI Q F,et al.Study on a prolyl endopeptidase from the skeletal muscle of common carp(Cyprinus carpio)[J].Process Biochemistry,2012,47(12):2211-2218.DOI:10.1016/j.procbio.2012.08.016.
[10]WU G P,CHEN S H,LIU G M,et al.Purification and characterization of a collagenolytic serine proteinase from the skeletal muscle of red sea bream(Pagrus major)[J].Comparative Biochemistry & Physiology Part B:Biochemistry & Molecular Biology,2010,58(9):5730-5736.
[11]LADRAT C,VERREZ-BAGNIS V,NO?L J,et al.Milli-Calpain fromsea bass(Dicentrarchus labrax) white muscle:purification,characterization of its activity and activation in vitro[J].Marine Biotechnology,2002,4(1):51-62.
[12]WU J L,GE S Y,CAI Z X,et al.Purification and characterization of a gelatinolytic matrix metalloproteinase from the skeletal muscle of grass carp(Ctenopharyngodon idellus)[J].Food Chemistry,2014,145(7):632-638.DOI:10.1016/j.foodchem.2013.08.055.
[13]肖琳琳,翁凌,钟婵,等.罗非鱼脯氨酸内肽酶的分离及抑制剂对其的作用机制[J].集美大学学报(自然科学版),2017,22(1):10-20.
[14]KUMAR R,BAVI R,JO M G,et al.New compounds identified through in silico approaches reduce the α-synuclein expression by inhibiting prolyl oligopeptidase in vitro[J].Scientific Reports,2017,7:1-14.DOI:10.1038/s41598-017-11302-0.
[15]李静,张剑,赵永祥.金属离子对蛋白酶作用的研究进展[J].日用化学工业,2017,47(6):345-351.
[16]刘成,韩宏岩,张颖,等.银离子对辣根过氧化物酶的影响[J].生物技术,2006(4):49-52.
[17]KURLAND L T.Amylotrophic lateral sclerosis and Parkinson's disease complex on Guam linked to an environmental neurotoxin[J].Trends in Neurosciences,1988,11(2):51-58.DOI:10.1016/0166-2236(88)90163- 4.
[18]SUMATHI T.Protective role of cynodon dactylon in ameliorating the aluminium-induced neurotoxicity in rat brain regions[J].Biological Trace Element Research,2011,144(1/3):843-853.DOI:10.1007/s12011-011-9029-6.
[19]卢斌,柯才焕,王文雄.低浓度镉、锌暴露对白氏文昌鱼的毒性累积及其几种重要酶活性的影响[J].厦门大学学报(自然科学版),2012,51(4):767-773.