Gilissen R A, Bamforth K J, Stavenuiter J F, Coughtrie M W, Meerman J H
Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands.
Carcinogenesis. 1994 Jan;15(1):39-45. doi: 10.1093/carcin/15.1.39.
Sulfation activity towards various heterocyclic and homocyclic aromatic hydroxamic acids and hydroxylamines was determined in adult human liver cytosol and with partially purified human liver sulfotransferases (STs). In adult human liver cytosols comparable ST activities towards N-hydroxy-2-acetyl-amino-5-phenylpyridine (N-OH-2AAPP), N-hydroxy-4-acetylaminobiphenyl (N-OH-4AABP) and N-hydroxy-4'fluoro-4-acetylaminobiphenyl (N-OH-4FAABP) were found, while the sulfation rates towards N-hydroxy-2-acetylaminofluorene (N-OH-2AAF), N-hydroxy-2-acetylaminonaphthalene (N-OH-2AAN), N-hydroxy-2-acetylaminophenanthrene (N-OH-2AAP) and N-hydroxy-4-acetylaminostilbene (N-OH-4AAS) were two- to five-fold lower. In adult liver cytosol ST activity was found towards all hydroxylamines tested. No significant differences were found for the various hydroxylamines. In general, the ST activities towards the various hydroxamic acids and hydroxylamines were comparable to phenol ST activity using adult liver cytosols. Partial purification of adult human liver STs was achieved by DEAE-Sepharose chromatography followed by anion exchange FPLC. Two separated protein peaks showing both N-OH-2AAPP and N-OH-2APP ST activities were observed and were designated human hydroxylamine/hydroxamic acid sulfotransferase (hHST) 1 and 2. Immunoblot analysis using an anti-rat estrogen ST antibody demonstrated cross reactivity with both hHSTs at a subunit mol. wt of 32 kDa corresponding to the phenol-sulfating form of phenol ST (P-PST). ST activity towards dopamine was low with both hHSTs, but hHST1 also contained significant capacity to sulfate dehydroepiandrosterone. The highest ST activity towards N-OH-2AAPP and N-OH-2APP was measured at pH 5.5 with both hHSTs. The Km values of the two hHSTs for sulfation of N-OH-2AAPP and N-OH-2APP were comparable, while the Vmax values for sulfation of N-OH-2APP were higher than for N-OH-2AAP with both hHSTs. FPLC anion exchange analysis of human platelet STs demonstrated that sulfation of N-OH-4ABP and N-OH-4AABP was associated with P-PST rather than M-PST (platelets do not possess any significant DHEA ST activity). Our results show that the various hydroxamic acids and hydroxylamines are converted by at least two hHSTs. The results presented here for the human liver hydroxamic acid and hydroxylamine ST activities are discussed in relation to those observed in the rat.
在成人肝细胞溶胶以及部分纯化的人肝脏磺基转移酶（STs）中，测定了对各种杂环和同环芳香族异羟肟酸及羟胺的硫酸化活性。在成人肝细胞溶胶中，发现对N - 羟基 - 2 - 乙酰氨基 - 5 - 苯基吡啶（N - OH - 2AAPP）、N - 羟基 - 4 - 乙酰氨基联苯（N - OH - 4AABP）和N - 羟基 - 4'-氟 - 4 - 乙酰氨基联苯（N - OH - 4FAABP）具有相当的ST活性，而对N - 羟基 - 2 - 乙酰氨基芴（N - OH - 2AAF）、N - 羟基 - 2 - 乙酰氨基萘（N - OH - 2AAN）、N - 羟基 - 2 - 乙酰氨基菲（N - OH - 2AAP）和N - 羟基 - 4 - 乙酰氨基茋（N - OH - 4AAS）的硫酸化速率低两到五倍。在成人肝细胞溶胶中发现对所有测试的羟胺都有ST活性。各种羟胺之间未发现显著差异。总体而言，使用成人肝细胞溶胶时，对各种异羟肟酸和羟胺的ST活性与苯酚ST活性相当。通过DEAE - 琼脂糖层析随后进行阴离子交换快速蛋白质液相色谱法，实现了成人肝脏STs的部分纯化。观察到两个分离的蛋白峰，它们都显示出N - OH - 2AAPP和N - OH - 2APP ST活性，并被命名为人羟胺/异羟肟酸磺基转移酶（hHST）1和2。使用抗大鼠雌激素ST抗体进行的免疫印迹分析表明，在32 kDa的亚基分子量处，两种hHST与苯酚ST（P - PST）的硫酸化苯酚形式具有交叉反应性。两种hHST对多巴胺的ST活性都很低，但hHST1也具有显著的硫酸化脱氢表雄酮的能力。两种hHST对N - OH - 2AAPP和N - OH - 2APP的最高ST活性在pH 5.5时测得。两种hHST对N - OH - 2AAPP和N - OH - 2APP硫酸化的Km值相当，而对N - OH - 2APP硫酸化的Vmax值高于对N - OH - 2AAP硫酸化的Vmax值。对人血小板STs的快速蛋白质液相色谱阴离子交换分析表明，N - OH - 4ABP和N - OH - 4AABP的硫酸化与P - PST相关，而不是与M - PST相关（血小板不具有任何显著的脱氢表雄酮ST活性）。我们的结果表明，各种异羟肟酸和羟胺至少由两种hHST进行转化。本文给出的关于人肝脏异羟肟酸和羟胺ST活性的结果与在大鼠中观察到的结果相关联进行了讨论。
