Use of antibodies to human O6-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O6-benzylguanine or N-methyl-N'-nitro-N-nitrosoguanidine.
Use of antibodies to human O6-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O6-benzylguanine or N-methyl-N'-nitro-N-nitrosoguanidine.
复制标题
使用人 O6-烷基鸟嘌呤-DNA 烷基转移酶抗体来研究经 O6-苄基鸟嘌呤或 N-甲基-N-硝基-N-亚硝基胍处理的细胞中该蛋白质的含量。
DOI：
10.1093/carcin/12.9.1679
复制
发表时间：
1991
期刊：
影响因子：
4.7
通讯作者：
Dolan,ME
中科院分区：
文献类型：
--
作者：
Pegg,AE;Wiest,L;Mummert,C;Stine,L;Moschel,RC;Dolan,ME
本文用抗人O_6-烷基鸟嘌呤-DNA烷基转移酶（O_6-alkylguanine-DNA alkyltransferase，O_6-benzylguanidine，O_6-alkylguanine-DNA alkyltransferase，O_6-alkylguanidine-DNA alkyltransferase）三种多肽的兔抗血清，研究了N-甲基-N′-硝基-N-亚硝基胍和O_6-苄基胍作用于人结肠癌细胞后，O_6-alkyltransferase蛋白在细胞在这些条件下，烷基转移酶蛋白失活，可能是由于其半胱氨酸受体位点转化为S-甲基半胱氨酸或S-苄基半胱氨酸。结果发现，在完整细胞和无细胞提取物中，这种失活后的蛋白质迅速降解。蛋白质的构象变化可能是通过半胱氨酸受体位点的烷基化将烷基转移酶转化为非活性形式而引起的。这种变化可能使蛋白质对蛋白水解降解非常敏感。无活性形式的蛋白质的快速降解可以作为其再合成的信号，但在短期内确保其通过半胱氨酸受体位点的再生而再活化不可能发生到任何显著的程度。灭活的烷基转移酶蛋白质的半衰期短，使得有可能通过免疫组织化学测量烷基转移酶蛋白质的含量，这可能是测量活性和灭活形式的蛋白质的总和，但仍会产生一个准确的估计细胞修复O 6-甲基鸟嘌呤的能力，提供了具有足够的特异性和亲和力的程序可以开发。
Antisera raised in rabbits to three peptides corresponding to amino acid sequences found in humanO6-alkylguanine-DNA alkyltransferase were used to study the fate of the alkyltransferase protein in human colon tumor cells after exposure toN-methyl-N′-nitro-N-ntrosoguanidine or toO6-benzylguanidine. Under these conditions, the alkyltransferase protein becomes inactivated, presumably by the conversion of its cysteine acceptor site to S-methylcysteine or S-benzylcysteine rrspectively. It was found that the protein was rapidly degraded after such inactivation both in intact cells and in cell-free extracts. It is probable that a conformational change in the protein is brought about by conversion of the alkyltransferase to the Inactive form by alkylation of the cysteine acceptor site. This change may render the protein very sensitive to proteolytic degradation. The rapid degradation of the inactive form of the protein may serve as a signal for its resynthesis but in the short term ensures that its reactivation by regeneration of the cysteine acceptor site is unlikely to occur to any sigificant extent. The short halflife of the inactivated alkyltranferase protein makes it probable that measurement of the content of the alkyltransferase protein by immunohistochemistry, which is likely to measure the sum of the active and inactivated forms of the protein, will nevertheless yield an accurate estimation of the cellular capacity to repairO6-methylguanine provided that procedures with sufficient specificity and affinity can be developed.