关闭
 
读者在线:用户名 密码
首页 期刊简介 投稿须知 期刊目录 专家风采 编委会 特邀顾问 广告发行 联系我们 移动出版
  1. 1
  2. 2
  3. 3
  4. 4
  5. 5



刊物信息

期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

访问统计
您是第  1 5 9 4 1 4 5 位浏览者
您当前的位置:首页 >> 正文

人红细胞内核苷二磷酸激酶的酶学特性及动力学研究

Kinetic and mechanistic characterizations of human erythrocytic nucleoside diphosphate kinase

作者(英文):
分类号:R917
出版年·卷·期(页码):2018,38 (3):385-392
DOI: 10.16155/j.0254-1793.2017.01.01
-----摘要:-------------------------------------------------------------------------------------------

目的:探讨人红细胞(RBC)内核苷二磷酸激酶(NDPK)的酶学动力学特性。方法:利用NDPK催化反应底物二磷酸脱氧腺苷、三磷酸脱氧鸟苷生成三磷酸脱氧腺苷及二磷酸脱氧鸟苷(dADP+dGTP↔dATP+dGDP)的酶促反应,通过改变反应温度、热处理温度、pH条件、底物浓度及抑制剂浓度进行NDPK酶学动力学研究。结果:底物动力学研究中,Linewever-Burk双倒数图为两组平行直线。产物抑制动力学研究中,固定反应底物dGTP浓度并改变dADP浓度时,dATP表现为非竞争性抑制作用,固定反应底物dADP浓度并改变dGTP浓度时,dATP表现为竞争性抑制作用。反应遵循乒乓机制。文中实验条件下,NDPK酶促反应最大速度Vmax约为110 μmol·min-1·g-1,KmdADP=1.26 mmol·L-1KmdGTP=1.94 mmol·L-1。dATP抑制常数Ki约为0.34~0.67 mmol·L-1结论:NDPK为酶促反应dADP+dGT dAPT+dGDP的一种高亲和性催化酶。

-----英文摘要:---------------------------------------------------------------------------------------

Objective: To investigate the characteristics of enzyme kinetics of NDPK in human erythrocytes.Methods: The enzymatic reaction of dADP+dGTP↔dATP+dGDP was catalyzed specifically by NDPK.Enzymatic characterizations were investigated by changing the reaction temperatures,heat-treatment temperatures,pH conditions,substrate concentrations and inhibitors concentrations,respectively,based on the above-mentioned reaction.Results: Two sets of parallel lines were obtained when plotting Linewever-Burk double-reciprocal plots in enzyme-substrate kinetic studies.A typical pattern of noncompetitive inhibition was presented when using dADP as the variable substrate and dGTP as the fixed substrate in the presence of various concentrations of dATP.Meanwhile,competitive inhibition was observed when dGTP was the variable substrate and dADP as the fixed substrate in product inhibition studies.The erythrocytic NDPK followed a "ping-pong" mechanism was reached.An approximate value of Vmax of 110 μmol·min-1·g-1,and KmdADP of 1.26 mmol·L-1,KmdGTP of 1.94 mmol·L-1 were obtained under the conditions in our experiment.Inhibition constant Ki for dATP was considered in the range of 0.34-0.67 mmol·L-1.Conclusion: NDPK is a high-affinity catalytic enzyme for enzg matic reaction of dADP+dGTP+dATP+dGDP.

-----参考文献:---------------------------------------------------------------------------------------
[1] BERINGER A,CITTERIO-QUENTIN A,OTERO RO,et al.Determination of inosine 5'-monophosphate dehydrogenase activity in red blood cells of thiopurine-treated patients using HPLC[J].J Chromatogr B Anal Technol Biomed Life Sci,2017,1044-1045(2):194
[2] SAHASRANAMAN S,HOWARD D,ROY S.Clinical pharmacology and pharmacogenetics of thiopurines[J].Eur J Clin Pharmacol,2008,64(8):753
[3] KARNER S,SHI S,FISCHER C,et al.Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate ainase activity in erythrocytes:novel targets for thiopurine therapy?[J].Drug Monit,2012,32(2):119
[4] SHIN JY,WEY M,UMUTESI HG,et al.Thiopurine prodrugs mediate immunosuppressive effects by interfering with Rac1 protein Function[J].J Biol Chem,2016,291(26):13699
[5] DEWIT O,STARKEL P,ROBLIN X.Thiopurine metabolism monitoring:implications in inflammatory bowel diseases[J].Eur J Clin Invest,2010,40(11):1037
[6] PRABHU VV,SIDDIKUZZAMAN,GRACE VM,et al.Targeting tumor metastasis by regulating Nm23 gene expression[J].Asian Pac J Cancer Prev,2012,13(8):3539
[7] TONG Y,YUNG LY,WONG YH.Metastasis suppressors Nm23H1 and Nm23H2 differentially regulate neoplastic transformation and tumorigenesis[J].Cancer Lett,2015,361(2):207
[8] BRAUNAGEL D,SCHAICH M,KRAMER M,et al.The T_T genotype within the NME1 promoter single nucleotide polymorphism-835 C/T is associated with an increased risk of cytarabine induced neurotoxicity in patients with acute myeloid leukemia[J].Leuk Lymphoma,2012,53(5):952
[9] VARGA A,GRACZER E,CHALOIN L,et al.Selectivity of kinases on the activation of tenofovir,an anti-HIV agent[J].Eur J Pharm Sci,2013,48(1-2):307
[10] GROSS S,DEVRAJ K,FENG Y,et al.Nucleoside diphosphate kinase B regulates angiogenic responses in the endothelium via caveolae formation and c-Src-mediated caveolin-1 phosphorylation[J].J Cereb Blood Flow Metab,2017,37(7):2471
[11] TEOH J,BOULOS S,CHIENG J,et al.Erythropoietin increases neuronal NDPKA expression,and NDPKA up-regulation as well as exogenous application protects cortical neurons from in vitro ischemia-related insults[J].Cell Mol Neurobiol,2014,34(3):379
[12] VELUTHAKAL R,KAETZEL D,KOWLURU A.Nm23-H1 regulates glucose-stimulated insulin secretion in pancreatic β-cells via Arf6-Rac1 signaling axis[J].Cell Physiol Biochem,2013,32(3):533
[13] BOSNAR MH,BAGO R,CETKOVIC H.Subcellular localization of nm23/NDPK A and B isoforms:a reflection of their biological function[J].Mol Cell Biochem,2009;329(1-2):63
[14] MOURAD N,PARKS RE.Erythrocytic nucleoside diphosphokinase Ⅱ.Isolation and kinetics[J].J Biol Chem,1966,241(2):271
[15] 沈如飞,刘亚妮,曾繁典,等.离子对高效液相色谱法测定人体红细胞内核苷二磷酸激酶活性[J].中国医院药学杂志,2012,32(4):252 SHEN RF, LIU YN,ZENG FD,et al.Determination of the activity of nucleoside diphosphate kinase in human red blood cell by reversed phase ion-pair HPLC[J].Chin Hosp Pharm J,2012,32(4):252
[16] LASCU I,GONIN P.The catalytic mechanism of nucleoside diphosphate kinases[J].J Bioenerg Biomembr,2000,32(3):215
[17] TAKACS-VELLAI K,VELLAI T,FARKAS Z,et al.Nucleoside diphosphate kinases (NDPKs) in animal development[J].Cell Mol Life Sci,2015,72(8):1447
[18] BOISSAN M,DABERNAT S,PEUCHANT E,et al.The mammalian Nm23/NDPK family:from metastasis control to cilia movement[J].Mol Cell Biochem,2009,329(1-2):51
[19] PERINA D,BOSNAR MH,BAGO R,et al.Sponge non-metastatic group I Nme gene/protein-structure and function is conserved from sponges to humans[J].BMC Evol Biol,2011,11(4):87
[20] ERENT M,GONIN P,CHERFILS J,et al.Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C,product of the DRnm23 gene[J].Eur J Biochem,2001,268(7):1972

欢迎阅读《药物分析杂志》!您是该文第 84位读者!

药物分析杂志 © 2009
地址:北京天坛西里2号
邮政编码:100050; 技术支持:010-60213898

电子邮件:ywfx@nicpbp.org.cn