Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli.
Effect of expression of human spermidine/spermine N1-acetyltransferase in Escherichia coli.
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人亚精胺/精胺 N1-乙酰转移酶在大肠杆菌中表达的影响。
DOI：
10.1021/bi00008a038
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Pegg,AE
中科院分区：
文献类型：
--
作者：
Parry,L;Lopez-Ballester,J;Wiest,L;Pegg,AE
摘要：为了在大肠杆菌中表达亚精胺/精胺N1-乙酰转移酶(SSAT)，构建了表达载体pINSAT2。用该载体转化的细胞产生了大量的SSAT，当加入异丙基/3-D-硫代半乳糖苷(IPTG)时，SSAT占可溶性蛋白的2%，在没有诱导剂的情况下，SSAT占可溶性蛋白的0.3%。表达SSAT的细胞生长速度减慢，细胞内的亚精胺全部转化为(V‘-乙酰亚精胺)，大部分亚精胺被排出。腐胺和1-甲基亚精胺(不是SSAT的底物)可以逆转SSAT表达对生长的影响，但亚精胺只有在通过省略IPTG诱导剂来限制SSAT的表达量时才有效。亚精胺缺乏对生长的刺激与其完全转化为A^-乙酰亚精胺有关。这些结果表明，A^-乙酰精胺不能替代未经修饰的多胺来支持生长，并提示乙酰化是一种生理反应，将多余的多胺转化为易于排泄的生理惰性形式。大量表达SSAT的细胞对抗肿瘤药物NL-IV12-双(乙基)精胺的生长抑制作用更为敏感，支持这种双(乙基)多胺诱导SSAT的能力有助于其抗增殖作用的假说。从含有pINSAT2的DH5a细胞提取液中，SSAT易于纯化至均一。纯化的酶与从人结肠癌细胞中纯化的精胺和亚精胺具有相似的比活力和效价，提示酶活性不需要对真核细胞进行翻译后修饰。重组SSAT被发现能够乙酰化药物15-脱氧精灵、2-[(氨丙基)氨基]乙硫醇和/V-(正丁基)-1，3-二氨基丙烷。精胺/精胺A^-乙酰基转移酶(SSAT)1是一种高度诱导的细胞溶血酶，首次从四氯化碳处理的大鼠肝脏的提取物中发现(Matsui et al.，1981)。后来的许多实验表明，SSAT是由多种刺激引起的，包括激素和生长因子、有毒化合物、各种药物和病理生理侮辱[Della Ragione等人(1984)和Casero和Pegg(1993)]。SSAT也由多胺本身和各种多胺类似物诱导(Casero&Pegg，1993；Persson&Pegg，1984；Pegg&Erwin，1985；Erwin&Pegg，1986；Casero等，；Saab等，1993；
Revised Manuscript Received December 16, 1994® abstract: A plasmid expression vector, pINSAT2, was constructed in order to express spermidine/spermine N1-acetyltransferase (SSAT) in Escherichia coli. Cellstransfected with this vector produced large amounts of SSAT, amounting to up to 2% of the soluble protein when isopropyl/3-D-thiogalactopyranoside (IPTG) was added and 0.3% of the soluble protein in the absence of inducer. The growth rate of cells expressing SSAT was reduced, and all of the cellular spermidine was converted to (V'-acetylspermidine, much of which was excreted. Putrescine and 1-methylspermidine, which is not a substrate for SSAT, could reverse the effects of SSAT expression on growth, but spermidine was only effective whenthe amount of SSAT expression was limited by omitting the IPTG inducer. The lack of stimulation of growth by spermidine correlated with its complete conversion to A^-acetylspermidine. These results show that A^-acetylspermine is not able to substitute for the unmodified polyamines in supporting growth and suggest thatacetylation is a physiological response to convert excess polyamines to a physiologically inert form which is readily excreted. Cells expressing large amounts of SSAT were much more senstive to the growth inhibitory action of the antitumor agent Nl, iV12-bis (ethyl) spermine, supporting the hypothesis that the ability of such bis (ethyl) polyamines to induce SSAT contributes to their antiproliferative actions. SSAT was readily purified to homogeneity from extracts of DH5a cells containing pINSAT2. The purified enzyme had a simialr specific activity and values for spermine and spermidine as the enzyme purified from human colon cancer cells, suggesting that posttranslational modifications specific to eukaryotes are not needed for enzymatic activity. The recombinant SSAT was found to acetylate the drugs 15-deoxyspergualin, 2-[(aminopropyl) amino] ethanethiol, and/V-(n-butyl)-1, 3-diaminopropane.Spermidine/spermine A^-acetyltransferase (SSAT) 1 is a highly inducible cytosolicenzyme that was first identified in extracts from the livers of rats treated with carbon tetrachloride (Matsui et al., 1981). Many subsequent experi-ments have shown thatSSAT is induced in responseto a wide range of stimuli including hormonesand growth factors, toxic compounds, various drugs, and pathophysiological insults [reviewed by Della Ragione et al.(1984) and Casero and Pegg (1993)]. SSAT isalso inducedby the polyamines themselves and by a variety of polyamine analogs (Casero & Pegg, 1993; Persson & Pegg, 1984; Pegg & Erwin, 1985; Erwin & Pegg, 1986; Casero et al., 1989; Saab et al., 1993;
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DOI：
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发表时间：
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期刊：
A M A Archives of Ophthalmology
影响因子：
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