Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay
Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay
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DOI：
10.1042/bj20061151
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发表时间：
2007-01-01
影响因子：
4.1
通讯作者：
Schofield, Christopher J.
中科院分区：
文献类型：
--
作者：
Ehrismann, Dominic;Flashman, Emily;Schofield, Christopher J.
后生动物HIT（缺氧诱导因子）的活性和水平受其羟基化调节，由2015（2-氧戊二酸盐）和Fe（II）依赖性双加氧酶催化。为了研究HIF羟化酶PHD2（脯氨酸羟化酶结构域蛋白2）和FIH（抑制HIF因子）重组形式的HIT羟化酶活性与氧浓度之间的关系，并与其他两种依赖于20g的双加氧酶进行了比较。虽然在绝对值上有一些注意事项，但PHD2和FIH的表观K（氧）值在其他2OG加氧酶观察到的范围内。与较短的HIF合成肽相比，重组蛋白底物具有较低的表观K（氧）值。分析还表明，人类PHD2对HIF-1 α的n端氧依赖性降解区域的c端片段具有选择性。目前的结果，尽管是在非生理条件下获得的，但意味着HIF羟化酶的表观K（氧）值使它们能够充当氧传感器，前提是它们的体内容量与羟化敏感信号通路适当匹配。
The activity and levels of the metazoan HIT (hypoxia-inducible factor) are regulated by its hydroxylation, catalysed by 2015 (2-oxoglutarate)- and Fe(II)-dependent dioxygenases. An oxygen consumption assay was developed and used to study the relationship between HIT hydroxylase activity and oxygen concentration for recombinant forms of two human HIF hydroxylases, PHD2 (prolyl hydroxylase domain-containing protein 2) and FIH (factor inhibiting HIF), and compared with two other 2OG-dependent dioxygenases. Although there are caveats on the absolute values, the apparent K (oxygen) values for PHD2 and FIH were within the range observed for other 2OG oxygenases. Recombinant protein substrates were found to have lower apparent K (oxygen) values compared with shorter synthetic peptides of HIF. The analyses also suggest that human PHD2 is selective for fragments of the C-terminal over the N-terminal oxygen-dependent degradation domain of HIF-1 alpha. The present results, albeit obtained under non-physiological conditions, imply that the apparent K (oxygen) values of the HIF hydroxylases enable them to act as oxygen sensors providing their in vivo capacity is appropriately matched to a hydroxylation-sensitive signalling pathway.