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Studies on Post-Translational Tyrosine Sulfation

翻译后酪氨酸硫酸化的研究

基本信息

批准号：
01560102
负责人：
SUIKO Masahito
金额：
$ 1.02万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

Sulfation in animal cells plays an important role in the metabolism of many exogenous and endogenous compounds such as phenol, catechol, steroid derivatives etc. And in post-translational modification of proteins on the tyrosine residue and on carbohydrate moieties. These reactions are catalyzed by distinct enzymes viz. Sulfotransferases, that transfer the sulfate moiety of 3'-Phospho-Adenosine-5'-Phosphosulfate (PAPS) to a hydroxyl group of an acceptor substrate. Various sulfotransferases have been demonstrated to be located in both the cytosol and membrane organelles of cells. However, it is now clear that post-translation tyrosine sulfation is catalyzed in most cases by membrane bound enzymes which are mostly located in the Golgi. This research was undertaken to study the Golgi located tyrosylprotein sulfotransferase in bovine liver and to develop an efficient system for tyrosine sulfation of recombinant proteins.Upon investigating the bovine liver microsomes we could identify a nov … More el membrane bound Phenol Sulfotransferase (PST) catalyzing sulfation of simple phenol derivatives, dopamine and tyramine, and showing a poor thermostability and what could be categorized as a "monoamine" form of phenol sulfotransferase.TPST has been characterized from the bovine liver Golgi membranes with activity on Z-Glu-Tyr, similar di and tri peptides and Boc-CCK-8. Among the substrates tested, peptides having a Asp on the N side of Tyr showed a higher reactivity than the peptides having a Glu at this position. Sulfation could not be detected on a peptide with a lysyl residue at the said position. Therefore it was postulated that substrate recognition of TPST, does depend on the amino acid sequence around the tyrosine residue.Hirudin, though has two Tyr residues is sulfated onl on the Tyr at position 63. However when hirudin is expressed in yeast, sulfation could not be observed on tyrosine 63 residue. Hence it is necessary to convert this recombinant product by in-vitro sulfation to its natural form. Sulfation of hirudin on the tyrosine 63 has been achieved with a heterologous TPST (bovine liver) with no sulfation on the tyrosine 3 residue. This confirmed that bovine liver TPST could selectively sulfate, tyrosine residues on specific amino acid sequences, in recombinant proteins. Less

动物细胞中的硫化作用在苯酚、邻苯二酚、类固醇衍生物等外源和内源化合物的代谢以及蛋白质对酪氨酸残基和碳水化合物部分的翻译后修饰中起着重要的作用。这些反应是由不同的酶催化的，即。硫基转移酶，将3‘-磷酸-腺苷-5’-磷酸(PAPS)的硫酸盐部分转移到受体底物的羟基上。已证实多种磺基转移酶存在于细胞的胞浆和膜细胞器中。然而，现在清楚的是，翻译后酪氨酸硫化在大多数情况下是由膜结合的酶催化的，这些酶主要位于高尔基体中。本研究旨在研究牛肝脏中高尔基体定位的酪氨酸蛋白磺基转移酶，并建立一种高效的重组蛋白酪氨酸硫酸盐化体系。通过对牛肝微粒体的研究，我们可以鉴定出一种新的…更多的EL膜结合的苯酚磺基转移酶(PST)催化简单的苯酚衍生物、多巴胺和酪胺的硫化，表现出较差的热稳定性，可以归类为苯酚磺基转移酶的“单胺”形式。在所测试的底物中，在Tyr的N侧具有天冬氨酸的多肽显示出比在该位置具有Glu的多肽更高的反应活性。在上述位置含有赖氨基残基的多肽上不能检测到硫酸盐化。因此推测TPST的底物识别确实依赖于酪氨酸残基周围的氨基酸序列。水飞蓟素虽然有两个Tyr残基，但在Tyr上只有63位是硫酸化的。然而，当水飞蓟素在酵母中表达时，不能观察到酪氨酸63残基的硫酸化。因此，有必要通过体外硫酸盐化将这种重组产物转化为其自然形式。利用异源TPST(牛肝)实现了水飞蓟素在酪氨酸63上的硫酸化，而不对酪氨酸3残基进行硫酸盐化。这证实了牛肝TPST在重组蛋白中可以选择性地将硫酸盐、酪氨酸残基定位在特定的氨基酸序列上。较少