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2. 发明申请

US20050054023A1 PEPTIDE SUBSTRATES FOR ASSAY OF EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASE 1 AND 2 ACTIVITY 失效
标题翻译：用于检测细胞外信号调节蛋白激酶1和2活性的肽基质
公开(公告)号：US20050054023A1
公开(公告)日：2005-03-10
申请号：US10126834
申请日：2002-04-19
申请人： John Haycock
发明人： John Haycock
IPC分类号： C12Q1/48 , C07K5/00 , C12N1/00 , C12N9/10
CPC分类号： C12Q1/485 , Y10S435/81
摘要： Several synthetic peptides modeled after Ser31 in tyrosine hydroxylase (“Ser31 peptides”) have been developed and evaluated as in vitro substrates for assaying the activity of extracellular signal-regulated protein kinase 1 and 2 (“ERK1/2”). The phosphorylation of the Ser31 peptides by activated, recombinant ERK2 was found to exhibit catalytic efficiencies (Vmax/Km) up to 4-fold higher than that of a synthetic myelin basic protein (MBP)-based peptide. Several synthetic peptides were tested using cellular extracts from PC 12 rat pheochromocytoma cells, both untreated cells and cells treated with nerve growth factor. Although the phosphorylation of the MBP peptide by extracts of PC12 cells was higher than that of the Ser31 peptide, the relative treatment-dependent increase was much greater for the Ser31 peptide and the pattern of ERK1/2 activation more closely mimicked the pattern seen with more complicated assays that initially isolated ERK1/2 from other kinases in the cellular extracts. This result suggested that the Ser31 peptide was a more specific substrate for the ERK1/2. Use of the new Ser31 peptide substrates will decrease the amount of peptide required to assay for ERK1/2 activity. In addition, the higher catalytic efficiencies associated with greater specificity for ERK1/2 will enable researchers to assay for activity of ERK1/2 in cellular extracts without prior immunoprecipitation.
摘要翻译：已经开发了几种在酪氨酸羟化酶（“Ser31肽”）Ser31之后建模的合成肽，并将其作为用于测定细胞外信号调节蛋白激酶1和2（“ERK1 / 2”）活性的体外底物。发现通过活化的重组ERK2的Ser31肽的磷酸化显示比基于合成髓磷脂碱性蛋白（MBP）的肽高达4倍的催化效率（Vmax / Km）。使用来自PC12大鼠嗜铬细胞瘤细胞（未处理的细胞）和用神经生长因子处理的细胞的细胞提取物测试几种合成肽。虽然PC12细胞提取物对MBP肽的磷酸化水平高于Ser31肽，但Ser31肽的相对治疗依赖性增加明显增加，ERK1 / 2激活模式更为模仿了更多的模式最初从细胞提取物中的其他激酶分离ERK1 / 2的复杂测定法。该结果表明Ser31肽是ERK1 / 2的更特异性底物。使用新的Ser31肽底物将降低测定ERK1 / 2活性所需的肽量。此外，与ERK1 / 2的更高特异性相关的更高的催化效率将使研究人员能够在没有事先免疫沉淀的情况下测定细胞提取物中ERK1 / 2的活性。

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