| 查看: 2774 | 回复: 6 | |||
| 本帖产生 1 个 翻译EPI ，点击这里进行查看 | |||
[交流]
急！帮忙翻译一些分子生物学方面的英文文献段落
|
|||
|
Based on the facts that purified ENTPD5 is unable to hydrolyze ATP directly and the assay also contained S-100 from PTEN heterozygous MEFs, we realized that there must be more factors in the S-100, which are also required to hydrolyze ATP to AMP. These factors presented in cells regardless of their PTEN status. For example, when we added purified, recombinant ENTPD5 and UMP to the dialyzed S-100 from large-scale cultured HeLa cells, the ATP-to-AMP hydrolysis was reconstituted (Figure 4A, lanes 1–6). This observation made purification of these factors easier because HeLa cells can be grown in large quantity insuspension. To identify these factors, we fractionated HeLa cell S-100, using a Q Sepharose column, and collected both the flowthrough (Q-FL) and column-bound fractions eluted with 300 mM NaCl (Q-30). Neither fraction alone was able to hydrolyze ATP to AMP, although the Q-30 fraction, when ENTPD5 and UMP were present, hydrolyzed ATP to ADP (Figure 4A, lanes 13 and 14). When both the Q-FL and Q-30 fractions were included, the ATP-to-AMP activity was fully reconstituted (Figure 4A, lane 18). We purified the activity present in the Q-30 fraction. The activity present in the Q-30 fraction was purified by subjecting HeLa S-100 onto four sequential column chromatographic steps and finally onto a Mini Q column (Figure 4B, left). The activity was eluted from this column with a linear salt gradient from 40 to 120 mM NaCl, and fractions eluted from the column were assayed in the presence of recombinant ENTPD5, UMP, and the Q-FL fraction (Figure 4B, right-bottom). A peak of activity was observed at fractions 8–10. The same fractions were subjected to SDS-PAGE followed by silver staining, and two protein bands close to 37 and 20 kDa markers correlated perfectly with activity (Figure 4B, right-top). Both bands were identified by mass spectrometry as human UMP/CMP kinase-1 (CMPK1).The identification of UMP/CMP kinase in the Q-30 fraction shed light on why UMP is a cofactor for the ATPase activity and how ENTPD5 plus this enzyme generates ADP from ATP. In this reaction, UMP is phosphorylated into UDP by CMPK1 and ATP, generating ADP. UDP is subsequently hydrolyzed by ENTPD5 to UMP, completing the cycle with net conversion of ATP to ADP. With this knowledge, wethen made an educated guess that the third protein factor present in the Q flowthrough fraction should be an adenylate kinase, which converts two ADP into one ATP and AMP, causing the ATP-to-AMP conversion seen in PTEN null cell extracts. To confirm this, we took theQflowthrough fraction and subjected it to a gel-filtration column and collected the fractions eluted from the column to assay for ATP-to-AMP hydrolysis in the presence of UMP, purified recombinant ENTPD5, and the Q-30 fraction that contains CMPK1. An ATP-to-AMP activity peak centered at fractions 17 and 18 was observed (Figure 4C, top). When these factions were subjected to western blotting analysis using an antibody against adenylate kinase-1 (AK1), the detected western blotting band correlated perfectly with the activity peak (Figure 4C, bottom). The correlation was maintained with additional chromatographic steps (data not shown). We subsequently generated recombinant CMPK1 and AK1 in bacteria and purified them to homogeneity (Figure 4D, lanes 9 and 12). Purified recombinant ENTPD5 expressed in insect cells runs as a triplet on an SDS-PAGE gel that could be shifted down to a doublet after treatment by PNGase F, indicating that ENTPD-5 is glycosylated (Figure 4D, lanes 10 and 11). These purified recombinant proteins allowed us to reconstitute the ATP-to-AMP hydrolysis cycle. Only when all three enzymes and UMP were present, efficient ATP-to-AMP conversion was observed (Figure 4D, lanes 1–8). |
回复此楼» 猜你喜欢
听说今天filecode变了
已经有39人回复
-
固定端突然变了，今天
已经有8人回复
-
filecode
已经有15人回复
-
基金中了
已经有8人回复
-
filecode
已经有11人回复
-
大家散了吧，后缀研究没有意义，别浪费时间了，过好目前的每一天，不要焦虑
已经有6人回复
-
一个有机合成实验室都需要哪些设备？
已经有12人回复
-
求各位大神看下
已经有33人回复
-
咨询面上基金
已经有7人回复
-
影响面上的因素
已经有11人回复
» 抢金币啦！回帖就可以得到:
-
祈福
+5/595
-
中科院招聘博士后及科研助理
+2/236
-
一边期待，一边忐忑：发榜前的科研人正在想什么？
+5/200
-
3-羟基丙腈（CAS：109-78-4）
+1/88
-
1,2-双(三甲基硅氧基)苯（CAS：5075-52-5）
+1/85
-
香港城市大学郑星课题组诚聘博士后 (生态学、环境科学、植物学、大气科学背景)
+1/83
-
具身智能机器人公司招聘硬件工程师岗位 预算100万内
+1/80
-
南京大学医工交叉方向薛璐璐团队招聘脂质纳米材料与RNA递送方向博士后1-2名
+1/73
-
坐标重庆 诚征男友
+1/63
-
高耐腐蚀性的镁合金微弧氧化工艺
+1/18
-
双一流高校招收博士（现代技术管理/环境技术经济管理）及硕士研究生（长期有效）
+1/14
-
《上海高薪》VS《中科院》
+1/11
-
澳大利亚 Murdoch University 全奖博士招生（3个名额）地质化工冶金领域
+1/10
-
瑞典林雪平大学 高峰课题组 诚聘 氮化物基无机材料方向 博士后1名
+1/7
-
福建师范大学海峡柔性电子学院-2027年9月入学博士-考核制-电催、能源
+1/7
-
香港理工大学殷骏课题组诚聘1 名博士后以及2 名博士生
+1/6
-
请大家抽空欣赏一下我们网站的配图
+1/5
-
高弯曲强度玻璃制品的特殊加工工艺
+1/4
-
北京师范大学（珠海校区）赵亚然副教授招收2027年电催化硕士/博士研究生
+1/3
-
致每一位等待国自然结果的科研人：我们想听听你的心声
+1/1
前世今生(金币+5):排版辛苦了！！！ 2010-11-25 14:15:51
|
等待老板中，帮楼主排个版，非中文单词数：600 Based on the facts that purified ENTPD5 is unable to hydrolyze ATP directly and the assay also contained S-100 from PTEN heterozygous MEFs, we realized that there must be more factors in the S-100, which are also required to hydrolyze ATP to AMP. These factors presented in cells regardless of their PTEN status. For example, when we added purified, recombinant ENTPD5 and UMP to the dialyzed S-100 from large-scale cultured HeLa cells, the ATP-to-AMP hydrolysis was reconstituted (Figure 4A, lanes 1–6). This observation made purification of these factors easier because HeLa cells can be grown in large quantity insuspension. To identify these factors, we fractionated HeLa cell S-100, using a Q Sepharose column, and collected both the flowthrough (Q-FL) and column-bound fractions eluted with 300 mM NaCl (Q-30). Neither fraction alone was able to hydrolyze ATP to AMP, although the Q-30 fraction, when ENTPD5 and UMP were present, hydrolyzed ATP to ADP (Figure 4A, lanes 13 and 14). When both the Q-FL and Q-30 fractions were included, the ATP-to-AMP activity was fully reconstituted (Figure 4A, lane 18). We purified the activity present in the Q-30 fraction. The activity present in the Q-30 fraction was purified by subjecting HeLa S-100 onto four sequential column chromatographic steps and finally onto a Mini Q column (Figure 4B, left). The activity was eluted from this column with a linear salt gradient from 40 to 120 mM NaCl, and fractions eluted from the column were assayed in the presence of recombinant ENTPD5, UMP, and the Q-FL fraction (Figure 4B, right-bottom). A peak of activity was observed at fractions 8–10. The same fractions were subjected to SDS-PAGE followed by silver staining, and two protein bands close to 37 and 20 kDa markers correlated perfectly with activity (Figure 4B, right-top). Both bands were identified by mass spectrometry as human UMP/CMP kinase-1 (CMPK1).The identification of UMP/CMP kinase in the Q-30 fraction shed light on why UMP is a cofactor for the ATPase activity and how ENTPD5 plus this enzyme generates ADP from ATP. In this reaction, UMP is phosphorylated into UDP by CMPK1 and ATP, generating ADP. UDP is subsequently hydrolyzed by ENTPD5 to UMP, completing the cycle with net conversion of ATP to ADP. With this knowledge, wethen made an educated guess that the third protein factor present in the Q flowthrough fraction should be an adenylate kinase, which converts two ADP into one ATP and AMP, causing the ATP-to-AMP conversion seen in PTEN null cell extracts. To confirm this, we took theQflowthrough fraction and subjected it to a gel-filtration column and collected the fractions eluted from the column to assay for ATP-to-AMP hydrolysis in the presence of UMP, purified recombinant ENTPD5, and the Q-30 fraction that contains CMPK1. An ATP-to-AMP activity peak centered at fractions 17 and 18 was observed (Figure 4C, top). When these factions were subjected to western blotting analysis using an antibody against adenylate kinase-1 (AK1), the detected western blotting band correlated perfectly with the activity peak (Figure 4C, bottom). The correlation was maintained with additional chromatographic steps (data not shown). We subsequently generated recombinant CMPK1 and AK1 in bacteria and purified them to homogeneity (Figure 4D, lanes 9 and 12). Purified recombinant ENTPD5 expressed in insect cells runs as a triplet on an SDS-PAGE gel that could be shifted down to a doublet after treatment by PNGase F, indicating that ENTPD-5 is glycosylated (Figure 4D, lanes 10 and 11). These purified recombinant proteins allowed us to reconstitute the ATP-to-AMP hydrolysis cycle. Only when all three enzymes and UMP were present, efficient ATP-to-AMP conversion was observed (Figure 4D, lanes 1–8). |
2楼2010-11-23 09:35:56
3楼2010-11-23 16:54:30
4楼2010-11-23 18:19:51
5楼2010-11-23 18:35:09
★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★
前世今生(金币+50, 翻译EPI+1):万分感谢！！！ 2010-11-25 14:15:08
cfk580713(金币+30):谢谢参与，辛苦了阿 2010-11-27 10:34:47
前世今生(金币+50, 翻译EPI+1):万分感谢！！！ 2010-11-25 14:15:08
cfk580713(金币+30):谢谢参与，辛苦了阿 2010-11-27 10:34:47
| 因为ENTPD5不能直接将ATP水解为AMP，实验结果表明在ENTPD5中加入杂合MEFs了PTEN的 S-100也不能直接将ATP水解为AMP，因此我们认为在将ATP水解为AMP的过程中，S-100中还需要其它影响因子。这些因子存在于细胞的PTEN态，却被我们忽视了。例如，当我们将纯化、重组后的ENTPD5和UMP加入到从大量组培的HeLa细胞中透析出来的S-100中后， ATP即可被水解为AMP (图4A, 1–6行)。该研究使得分离这些影响因子变简单了，因为培养HeLa细胞很容易。为了确定这些因子，我们将HeLa细胞S-100用琼脂糖Q进行分段, 收集通柱流出液，记为Q-FL ；用 300 mM NaCl冲柱，收集流出液，记为Q-30.两部分均不能将ATP水解为AMP，但是Q-30在ENTPD5 和UMP的存在下，能将ATP水解为ADP。当Q-FL和 Q-30共同存在时，ATP可以水解为AMP(图 4A, 18行)。我们从Q-30段分离得到了影响因子。将Q-30依次通过4根色谱柱，最后再通过一个微型Q柱 (左图 4B)。 Q柱用40到120 mM NaCl进行梯度洗脱并收集，每部分用ENTPD5、UMP混合液和 Q-FL段进行试验 (右下，图 4B)。目标物峰值出现在8–10部分. 该部分通过 SDS-PAGE silver staining, 两蛋白质条带接近 marker 37 和20 kDa 与活性极相关(右上图4B)。经质谱鉴定为人 UMP/CMP激酶-1 (CMPK1)。 Q-30he中UMP/CMP激酶的发现 为我们解释了为什么UMP是ATP酶活性的影响因子和ENTPD5是怎样和这种酶共同作用将ATP水解为ADP的。在这个反应中， UMP在CMPK1和ATP 的作用下将UDP磷酸化，产生ADP。 UDP在ENTPD5的作用下被水解为UMP，完成ATP到ADP的一个循环。在这个基础上，我们推测第三个蛋白因子存在于Q-FL中，并且是可以将两个ADP转化为一个ATP和一个AM的腺苷酸激酶，使得PTEN零细胞提取物中有 ATP转化为AMP的过程。为证明该推论，我们对Q-FL进行了凝胶过滤， 收集滤液在UMP、纯化、重组的ENTPD5 以及含有CMPK1的Q-30部分的条件下进行试验检测 ATP转化AMP的活性。 ATP转化为AMP活性峰值集中于 17-18 段 (上图 4C)。将这两段用western杂交进行分析，用腺苷酸激酶-1 (AK1)抗体检测, 检测到的条带与活性峰完全相关 (下图 4C)。经其他色谱检测相关性依然存在 (无数据)。我们用细菌得到重组CMPK1和AK1蛋白并将他们进行了同质分离 (图 4D， 9-12行)。用SDS-PAGE检测显示，纯化后的重组 ENTPD5是寄主细菌细胞的3倍，用PNG酶 F处理过的重组 ENTPD5的表达是寄主的2倍，表明ENTPD-5被糖基化了(图4D，10-11行)。 这些重组蛋白让我们重现了 ATP到AMP的水解过程。只有当三种酶和UMP同时存在时， ATP转化为AMP的高效过程才能进行 (图4D, 1–8行). |
6楼2010-11-24 19:38:37
a俺要币币！ 7楼2010-11-24 19:40:11
