Effect of N-glycan removal on the enzymatic activity of porcine thyroid peroxidase.
Effect of N-glycan removal on the enzymatic activity of porcine thyroid peroxidase.
复制标题
N-聚糖去除对猪甲状腺过氧化物酶酶活性的影响。
DOI：
10.1111/j.1432-1033.1991.tb16401.x
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
A. Giraud
中科院分区：
文献类型：
--
作者：
Y. Long;J. Franc;J. Kaniewski;J. Lanet;A. Giraud
猪甲状腺过氧化物酶（pTPO）经脱氧胆酸盐抽提后免疫亲和层析（pTPO A）或胰蛋白酶/毛地黄皂苷抽提后离子交换凝胶过滤层析（pTPO B）纯化，pTPO A为全长分子，pTPO B为肽段。纯化的pTPO通过肽N-糖苷酶F（PNGase F）或通过内切-β-N-乙酰氨基葡糖苷酶H（endo H）处理去糖基化。电泳对照和伴刀豆球蛋白A的亲和印迹表明，去糖基化不是完全的，并且pTPO被endo H比被PNGase F更有效地去糖基化。通过愈创木酚和碘化物氧化检测的pTPO A的酶活性被PNGase F和endo H去糖基化抑制，而pTPO B的酶活性不受抑制。去糖基化后，pTPO A对愈创木酚和碘化物的表观Km增加，而对两种底物的Vmax降低。通过蔗糖密度梯度离心检查去糖基化前后pTPO A的聚集状态。结果表明，这种抑制不是由于pTPO A溶解度的损失。这些观察结果表明，去糖基化诱导的三级结构的pTPO A的影响的活性位点域的酶的修改。
Active porcine thyroid peroxidase (pTPO) has been purified either by deoxycholate extraction followed by immunoaffinity purification (pTPO A) or by trypsin/digitonin extraction followed by ion-exchange and gelfiltration chromatography (pTPO B); pTPO A appeared as a full-length molecule, while pTPO B appeared as peptide fragments. Purified pTPO were deglycosylated either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H) treatment. Electrophoretic controls and affinity blotting with concanavalin A indicated that deglycosylation was not total and that pTPO was more efficiently deglycosylated by endo H than by PNGase F. The enzymatic activity of pTPO A, checked by guaiacol and iodide oxidation, was inhibited by PNGase F and endo H deglycosylation, while that of pTPO B was not. After deglycosylation, the apparent Km of pTPO A for guaiacol and iodide increased, while the Vmax for both substrates decreased. The state of aggregation of pTPO A before and after deglycosylation was checked by sucrose density-gradient centrifugation. Results indicated that this inhibition was not due to a loss of pTPO A solubility. These observations suggest that deglycosylation induced a modification of the tertiary structure of pTPO A which affected the active-site domain of the enzyme.
猪甲状腺过氧化物酶结构基因的分子克隆。
DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Magnusson,RP;Gestautas,J;Taurog,A;Rapoport,B
通讯作者：
Rapoport,B