| 查看: 2440 | 回复: 6 | |||
| 本帖产生 1 个 翻译EPI ，点击这里进行查看 | |||
| 当前只显示满足指定条件的回帖，点击这里查看本话题的所有回帖 | |||
[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
求助:我三月中下旬出站，青基依托单位怎么办？
已经有9人回复
-
Cas 72-43-5需要30g，定制合成，能接单的留言
已经有8人回复
-
北京211副教授，35岁，想重新出发，去国外做博后，怎么样？
已经有8人回复
-
磺酰氟产物，毕不了业了！
已经有5人回复
-
论文终于录用啦！满足毕业条件了
已经有25人回复
-
2026年机械制造与材料应用国际会议 （ICMMMA 2026）
已经有3人回复
-
自荐读博
已经有3人回复
-
不自信的我
已经有5人回复
-
投稿Elsevier的杂志（返修），总是在选择OA和subscription界面被踢皮球
已经有8人回复
» 抢金币啦！回帖就可以得到:
-
博士去军队文职怎么样
+5/270
-
上海交通大学医学院王戈林课题组招聘博士后和助理研究员
+1/269
-
江汉大学招聘AI for Materials/电解液/锂金属/全固态电池等方面的博士或者博士后
+1/170
-
福建师范大学柔性电子学院招收2026年博士（储能材料与柔性电子器件）
+1/84
-
澳门大学智慧城市物联网国重“结构智能感知、健康监测与无损检测”研究方向博士后招聘
+1/79
-
坐标济南，山东农科院招 有机合成 or 药物化学 联培硕士研究生
+1/39
-
华中科技大学龚江研究员课题组诚招博士研究生、科研助理和博士后
+2/32
-
中国科学院大连化学物理研究所DNL0902研究组招聘博士后和职工
+1/32
-
厦门大学航空航天学院智能制造课题组招2026年申请审核制博士生1-2名
+1/31
-
北京林业大学木质素高值化利用创新团队招收2026年入学博士生
+1/26
-
可以用同一个研究内容申请青C和博士后面上吗
+1/25
-
征女友 @长安
+1/22
-
华中科技大学袁书珊教授团队招2026年申请审核制博士生1-2名
+1/22
-
杨老师招收联合培养硕士、博士生或客座学生
+1/19
-
SCI，计算机相关可以写
+1/14
-
深容SCI智能体四大模块：Method, Introduction, Discussion, Abstract
+1/3
-
电子科技大学激光与光子制造团队招硕士博士
+1/3
-
中国矿业大学黄赳课题组联合中国科学院南京土壤研究所朱晓芳研究员诚聘博士后
+1/2
-
2026 博士自荐-机器人机构学方向
+1/2
-
土木、交通工程专业博士后站有吗？（无博士毕业3年要求+可接受兼职博后）
+1/1
4楼2010-11-23 18:19:51
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
5楼2010-11-23 18:35:09