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Mass Spectrometric Analysis of Specificity and Inhibition of Group IVA Cytosolic Phospholipase A2 on Natural Membrane Phospholipids.

机译:质谱分析IVA胞质磷脂酶A2对天然膜磷脂的特异性和抑制作用。

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摘要

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that is well-studied by the scientific community. PLA2s are membrane-associated enzymes that catalyze the hydrolysis of the ester bond at the sn-2 position of membrane phospholipids liberating free fatty acids, predominantly arachidonic acid (AA). The PLA 2 superfamily consists of 16 groups and numerous subgroups and they can be considered as six major types: the secreted (sPLA2), the cytosolic (cPLA2), the calcium-independent (iPLA2), the platelet-activating factor acetylhydrolase (PAF-AH), lysosomal PLA 2 (LPLA2), and the adipose-PLA (AdPLA).;The human Group IVA cPLA2 (GIVA cPLA2) was cloned and sequenced from U937 cells in 1999. This enzyme contains 749 amino acids and has a molecular weight of 85.2 kDa. The crystal structure of the enzyme was resolved in 1999 and was found to contain an N-terminal C2 domain and a C-terminal catalytic domain. GIVA cPLA2 is of important biological relevance and involved at the earliest stage of inflammation since through its specificity for AA at the sn-2 position, it is the main AA provider for the eicosanoid biosynthetic pathway. Understanding the function of the enzyme is vital to find new ways to modulate inflammatory diseases in contrast to inhibiting traditional downstream pathways.;In this study, we have designed a liquid chromatography/mass spectrometry (LC/MS) high-throughput assay to test the specificity of GIVA cPLA2 toward different substrates. We tested various natural phospholipid substrates and found a preference for GIVA cPLA2 for 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine (PAPC). We also validated the assay for determining inhibitors constants and potency using commercially available inhibitors.
机译:磷脂酶A2(PLA2)构成了酶的一个超家族,受到科学界的广泛研究。 PLA2是与膜相关的酶,可催化膜磷脂的sn-2位置处的酯键水解,从而释放出游离脂肪酸,主要是花生四烯酸(AA)。 PLA 2超家族由16个亚类和许多亚类组成,它们可被认为是六种主要类型:分泌型(sPLA2),胞质(cPLA2),非钙依赖性(iPLA2),血小板活化因子乙酰水解酶(PAF- AH),溶酶体PLA 2(LPLA2)和脂肪PLA(AdPLA)。1999年从U937细胞克隆并测序了人类IVA cPLA2组(GIVA cPLA2)。该酶包含749个氨基酸,分子量为85.2 kDa。该酶的晶体结构于1999年解析,发现包含N端C2域和C端催化域。 GIVA cPLA2具有重要的生物学意义,并参与了炎症的最早阶段,因为通过其对sn-2位氨基酸的特异性,它是类花生酸生物合成途径的主要氨基酸提供者。与抑制传统的下游途径相反,了解酶的功能对于找到调节炎症性疾病的新方法至关重要。在本研究中,我们设计了一种液相色谱/质谱(LC / MS)高通量测定法来测试这种酶。 GIVA cPLA2对不同底物的特异性。我们测试了各种天然磷脂底物,发现偏爱GIVA cPLA2的是1-棕榈酰-2-花生四烯酰基-sn-甘油-3-磷酸胆碱(PAPC)。我们还验证了使用市售抑制剂测定抑制剂常数和效力的测定方法。

著录项

  • 作者

    Chen, Yuan.;

  • 作者单位

    University of California, San Diego.;

  • 授予单位 University of California, San Diego.;
  • 学科 Biochemistry.;Pharmacology.;Chemical engineering.
  • 学位 M.S.
  • 年度 2015
  • 页码 86 p.
  • 总页数 86
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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