一种改良的偶联酶法用于 O-连接的 N-乙酰葡萄糖胺转移酶活性测定。
A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay.
出版信息
Biol Proced Online. 2009 Dec 3;11:170-83. doi: 10.1007/s12575-009-9016-x.
Abstract
In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5'-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (E(m) = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT.
摘要
为了确定 O-连接的 N-乙酰氨基葡萄糖转移酶（OGT）的活性，提出了一种改良的偶联酶法。该方法基于转糖苷反应生成的产物尿苷 5'-(三磷酸氢)（UDP）的测量。在测定中，使用丙酮酸激酶将 UDP 与磷酸烯醇丙酮酸转化为丙酮酸偶联。使用商业丙酮酸测定试剂盒，将丙酮酸转化为红色末端产物，可在微孔板读数器上在 570nm 处进行比色测量或在 587nm（E(m) = 535nm）处进行荧光测量。对截断重组 mOGT 的动力学研究和两种生物样品中 OGT 的定量分析表明，该方法在定量分析 OGT 方面是实用和有竞争力的。
相似文献
1
A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay.一种改良的偶联酶法用于 O-连接的 N-乙酰葡萄糖胺转移酶活性测定。
Biol Proced Online. 2009 Dec 3;11:170-83. doi: 10.1007/s12575-009-9016-x.
2
New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro.基于酶联免疫吸附测定（ELISA）的体外检测O-连接N-乙酰葡糖胺转移酶活性的新方法。
Prep Biochem Biotechnol. 2017 Aug 9;47(7):699-702. doi: 10.1080/10826068.2017.1303614. Epub 2017 Mar 15.
3
Enhanced transfer of a photocross-linking N-acetylglucosamine (GlcNAc) analog by an O-GlcNAc transferase mutant with converted substrate specificity.具有转换底物特异性的O-连接N-乙酰葡糖胺（GlcNAc）转移酶突变体增强光交联N-乙酰葡糖胺类似物的转移。
J Biol Chem. 2015 Sep 11;290(37):22638-48. doi: 10.1074/jbc.M115.667006. Epub 2015 Aug 3.
4
Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats.一种胞质和核内O-连接N-乙酰葡糖胺转移酶的调控。四肽重复序列的作用。
J Biol Chem. 1999 Nov 5;274(45):32015-22. doi: 10.1074/jbc.274.45.32015.
5
Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity.O-连接的N-乙酰葡糖胺转移酶的功能表达。结构域结构和底物特异性。
J Biol Chem. 2000 Apr 14;275(15):10983-8. doi: 10.1074/jbc.275.15.10983.
6
Antibodies and activity measurements for the detection of O-GlcNAc transferase and assay of its substrate, UDP-GlcNAc.用于检测O-连接的N-乙酰葡糖胺转移酶及其底物UDP-N-乙酰葡糖胺的抗体和活性测量。
Methods Mol Biol. 2013;1022:147-59. doi: 10.1007/978-1-62703-465-4_12.
7
Single Quantum Dot-Based Nanosensor for Sensitive Detection of O-GlcNAc Transferase Activity.基于单量子点的纳米传感器用于灵敏检测 O-GlcNAc 转移酶活性。
Anal Chem. 2017 Dec 5;89(23):12992-12999. doi: 10.1021/acs.analchem.7b04065. Epub 2017 Nov 17.
8
Mechanism of pyruvate-uridine diphospho-N-acetylglucosamine transferase. Evidence for an enzyme-enolpyruvate intermediate.丙酮酸-尿苷二磷酸-N-乙酰葡糖胺转移酶的作用机制。酶-烯醇丙酮酸中间体的证据。
J Biol Chem. 1975 Jul 10;250(13):4959-64.
9
Uridine diphosphate release mechanism in O-N-acetylglucosamine (O-GlcNAc) transferase catalysis.在 O-N-乙酰葡萄糖胺（O-GlcNAc）转移酶催化中尿苷二磷酸的释放机制。
Biochim Biophys Acta Gen Subj. 2019 Mar;1863(3):609-622. doi: 10.1016/j.bbagen.2018.12.005. Epub 2018 Dec 11.
10
Measurement of UDP-N-acetylglucosaminyl transferase (OGT) in brain cytosol and characterization of anti-OGT antibodies.脑细胞质中UDP-N-乙酰葡糖胺基转移酶（OGT）的测定及抗OGT抗体的特性分析
Anal Biochem. 2003 Mar 15;314(2):169-79. doi: 10.1016/s0003-2697(02)00686-3.
引用本文的文献
1
-GlcNAcylation ameliorates the pathological manifestations of Alzheimer's disease by inhibiting necroptosis.N-乙酰葡糖胺化通过抑制坏死性凋亡改善阿尔茨海默病的病理表现。
Sci Adv. 2021 Jan 13;7(3). doi: 10.1126/sciadv.abd3207. Print 2021 Jan.
2
O-GlcNAcylation of G6PD promotes the pentose phosphate pathway and tumor growth.葡萄糖-6-磷酸脱氢酶的O-连接N-乙酰葡糖胺化促进磷酸戊糖途径和肿瘤生长。
Nat Commun. 2015 Sep 24;6:8468. doi: 10.1038/ncomms9468.
本文引用的文献
1
Triclosan resistance in Salmonella enterica serovar Typhimurium.肠炎沙门氏菌鼠伤寒血清型对三氯生的耐药性。
J Antimicrob Chemother. 2008 Jul;62(1):83-91. doi: 10.1093/jac/dkn137. Epub 2008 Apr 3.
2
Aging leads to increased levels of protein O-linked N-acetylglucosamine in heart, aorta, brain and skeletal muscle in Brown-Norway rats.衰老导致棕色挪威大鼠心脏、主动脉、大脑和骨骼肌中O-连接的N-乙酰葡糖胺水平升高。
Biogerontology. 2008 Jun;9(3):139. doi: 10.1007/s10522-007-9123-5. Epub 2008 Jan 10.
3
A cellular FRET-based sensor for beta-O-GlcNAc, a dynamic carbohydrate modification involved in signaling.一种基于细胞荧光共振能量转移的β-O-连接的N-乙酰葡糖胺传感器，β-O-连接的N-乙酰葡糖胺是一种参与信号传导的动态碳水化合物修饰。
J Am Chem Soc. 2006 Nov 22;128(46):14768-9. doi: 10.1021/ja065835+.
4
O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.使用凝集素弱亲和色谱法和质谱法对突触后致密体制剂进行O-连接的N-乙酰葡糖胺蛋白质组学研究。
Mol Cell Proteomics. 2006 May;5(5):923-34. doi: 10.1074/mcp.T500040-MCP200. Epub 2006 Feb 1.
5
Recombinant O-GlcNAc transferase isoforms: identification of O-GlcNAcase, yes tyrosine kinase, and tau as isoform-specific substrates.重组O-连接N-乙酰葡糖胺转移酶同工型：鉴定O-连接N-乙酰葡糖胺酶、Yes酪氨酸激酶和tau作为同工型特异性底物。
Glycobiology. 2006 May;16(5):415-21. doi: 10.1093/glycob/cwj078. Epub 2006 Jan 23.
6
Measurement of UDP-N-acetylglucosaminyl transferase (OGT) in brain cytosol and characterization of anti-OGT antibodies.脑细胞质中UDP-N-乙酰葡糖胺基转移酶（OGT）的测定及抗OGT抗体的特性分析
Anal Biochem. 2003 Mar 15;314(2):169-79. doi: 10.1016/s0003-2697(02)00686-3.
7
Mitochondrial and nucleocytoplasmic targeting of O-linked GlcNAc transferase.O-连接的N-乙酰葡糖胺转移酶的线粒体和核质靶向
J Cell Sci. 2003 Feb 15;116(Pt 4):647-54. doi: 10.1242/jcs.00246.
8
Identification of GlcNAcylation sites of peptides and alpha-crystallin using Q-TOF mass spectrometry.
J Am Soc Mass Spectrom. 2001 Oct;12(10):1106-13. doi: 10.1016/s1044-0305(01)00295-1.
9
Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats.一种胞质和核内O-连接N-乙酰葡糖胺转移酶的调控。四肽重复序列的作用。
J Biol Chem. 1999 Nov 5;274(45):32015-22. doi: 10.1074/jbc.274.45.32015.
10
O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats.O-连接的N-乙酰葡糖胺转移酶是一种含有四肽重复序列的保守核质蛋白。
J Biol Chem. 1997 Apr 4;272(14):9316-24. doi: 10.1074/jbc.272.14.9316.
Zotero 插件