Heterodimeric deoxynucleoside kinases of Lactobacillus acidophilus R-26: functional assignment of subunits using limited proteolysis controlled by end-product inhibitors.
Heterodimeric deoxynucleoside kinases of Lactobacillus acidophilus R-26: functional assignment of subunits using limited proteolysis controlled by end-product inhibitors.
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嗜酸乳杆菌 R-26 的异二聚脱氧核苷激酶：使用由终产物抑制剂控制的有限蛋白水解对亚基进行功能分配。
DOI：
10.1021/bi00183a041
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Ives,DH
中科院分区：
文献类型：
--
作者：
Ikeda,S;Ma,GT;Ives,DH
摘要：嗜酸乳杆菌R-26的两种酶复合物具有脱氧胞苷激酶/脱氧腺苷激酶（Ⅰ）和脱氧鸟苷激酶/脱氧腺苷激酶（Ⅱ）活性，其四级结构已通过以下步骤得到证实：（1）在pH 6.6的SDS-PAGE上将每种复合物分离成两种组分;（2）每种组分的N-末端氨基酸序列测定;（3）通过差异限制性蛋白酶解对各组分进行功能定位。第三步是由以下发现促进的：特定终产物抑制剂dNTP与每个激酶活性位点的结合使相应的激酶亚基对胰蛋白酶具有抗性，同时使异源激酶亚基对蛋白水解敏感。SDS-PAGE分析显示，在dATP存在下，用胰蛋白酶对dCyd激酶/dAdo激酶（I）进行蛋白水解后，只有两个片段（15.8和11.0 kDa）。这可能表明，不受dNTP保护的激酶多肽链（27.2 kDa）在单个特定位点被胰蛋白酶切割，伴随着活性的丧失。因此，这项工作提出了一个独特的方法来澄清的结构和功能的酶组成的异源亚基。
Revised Manuscript Received February 25, 1994• abstract: Heterodimeric quaternary structures for two enzyme complexes fromLactobacillus acidophilus R-26 exhibiting deoxycytidine kinase/deoxyadenosine kinase (I) and deoxyguanosine kinase/deoxyadenosine kinase (II) activities have been proven by the following steps:(1) separation of each complex into two components on SDS-PAGE at pH 6.6;(2) N-terminal amino acid sequencing of each component;(3) functional assignment of each component by differential limited proteolysis. The third step was facilitated by the finding that the binding of a specific end-product inhibitor, dNTP, to each kinase active site makes the corresponding kinase subunit resistant to trypsin, while leaving the heterologous kinase subunit susceptible to proteolysis. Analysis on SDS-PAGE has revealed only two fragments (15.8 and 11.0 kDa) following proteolysis of dCyd kinase/dAdo kinase (I) with trypsin in the presence of dATP. This may indicate that the kinase polypeptide chain (27.2 kDa) not protected by dNTP is cut by trypsin at a single specific site, with concomitant loss of activity. Thus, this work presents a unique approach to the clarification of structure and function of enzymes composed of heterologous subunits.