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前世今生

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[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).

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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).

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6楼2010-11-24 19:38:37

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foryever

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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).

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2楼2010-11-23 09:35:56

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ringzhu

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引用回帖:

Originally posted by foryever at 2010-11-23 09:35:56:
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 realize ...

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