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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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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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