In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase
In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase
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DOI：
10.1016/j.biocel.2004.02.014
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发表时间：
2004-10-01
影响因子：
4
通讯作者：
Ikura, K
中科院分区：
文献类型：
--
作者：
Ichikawa, A;Ohashi, Y;Ikura, K
转氨酶催化蛋白质的交联和胺掺入，并且涉及各种生物现象。为了在分子水平上阐明转氨酶的生理作用，我们需要确定其生理蛋白底物，并阐明转氨酶修饰蛋白底物与生物学反应之间的关系。在这里，我们研究了甜菜碱-同型半胱氨酸S-甲基转移酶（BHMT：EC 2.1.1.5）是否可以在体外实验中使用猪肝BHMT和豚鼠肝转氨酶的组织型转氨酶的底物。豚鼠肝转氨酶以时间依赖性方式将5-（生物素酰胺基）戊胺和[3 H]组胺掺入BHMT。腐胺和亚精胺似乎也被纳入BHMT的转氨酶。在没有伯胺的情况下，BHMT亚基在分子内和分子间交联。通过转氨酶交联，BHMT活性显著降低。组胺掺入轻微降低BHMT活性。通过生物素标记、蛋白酶消化、生物素-亲和素a亲和分离和反相HPLC分离含有谷氨酰胺残基的BHMT肽片段，所述谷氨酰胺残基对转氨酶反应具有反应性。氨基酸序列分析及与其它哺乳动物肝脏BHMT亚基的同源性比对结果表明，这些活性谷氨酰胺残基位于猪BHMT亚基的羧基端附近。这些结果表明，肝BHMT可以被组织型转氨酶修饰，并且其活性受到修饰的抑制性调节，尤其是通过交联。这种调节反应可能参与了肝脏同型半胱氨酸代谢的调节。(C)2004爱思唯尔有限公司保留所有权利。
Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutaminase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [3 H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver. (C) 2004 Elsevier Ltd. All rights reserved.