RNA degradation in perfused rat liver as determined from the release of [14C]cytidine.
RNA degradation in perfused rat liver as determined from the release of [14C]cytidine.
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DOI：
10.1016/s0021-9258(18)47824-6
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发表时间：
1987-10
期刊：
影响因子：
--
通讯作者：
B. Lardeux;S. Heydrick;G. E. Mortimore
中科院分区：
文献类型：
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作者：
B. Lardeux;S. Heydrick;G. E. Mortimore
在循环灌流的大鼠肝脏中，通过释放标记的胞苷来确定RNA在循环灌流的大鼠肝脏中的降解，该RNA先前已被[6-14C]罗丹酸标记。由于胞苷在大鼠肝脏中没有明显的降解作用(它对尿苷的脱氨基几乎为零)，或者从游离的5‘-核苷酸中大量产生胞苷，它的释放将直接反映净RNA的分解。这一结论得到了以下事实的证实：在灌流180分钟后，释放的胞苷的比放射性等于RNA中的cMP的比放射性，并且保持不变。[~(14)C]胞苷的初始积累速率较慢，但在10-20分钟后突然增加4倍以上，并基本保持不变。添加0.5 mM的未标记胞苷有效地阻止了标记的重复利用，并使标记胞苷的释放量增加了13%，相当于最大胞苷积累速率的13%。在0.5 mM未标记胞苷存在下，20至60分钟的RNA降解速率平均为1.00+/-0.05 mg h-1肝-1(100-g大鼠)，相当于每天总RNA的65%。这个加速值大约是初始速率的4倍，被认为是氨基酸缺乏的直接结果，因为在单独的实验中，这种增加完全被血浆氨基酸的添加所抑制(Lardeux，B.R.和Mortimore，G.E.(1987)J.Biol)。化学。262、14514-14519)。这些发现证明了胞苷作为一种标记物的潜在价值，可用于在分离的肝脏或肝细胞制剂中跟踪RNA降解的时刻到时刻的调节变化。
The degradation of RNA in the cyclically perfused rat liver was determined from the release of labeled cytidine from RNA that had been previously labeled with [6-14C]orotic acid in vivo. Because cytidine is not appreciably degraded in rat liver (its deamination to uridine is virtually nil) or produced in significant amounts from free 5'-nucleotides, its release will directly reflect net RNA breakdown. This conclusion was substantiated by the fact that the specific radioactivity of released cytidine equaled that of CMP in RNA and remained unchanged for 180 min of perfusion. The initial rate of [14C]cytidine accumulation was slow, but after 10-20 min it increased abruptly by more than 4-fold and remained virtually constant. The addition of 0.5 mM unlabeled cytidine effectively prevented the reutilization of label and increased the rate of labeled cytidine release by an amount representing 13% of the maximal rate of cytidine accumulation. Rates of RNA degradation, measured between 20 and 60 min in the presence of 0.5 mM unlabeled cytidine, averaged 1.00 +/- 0.05 mg h-1 liver-1 (100-g rat), the equivalent of 65% of total RNA per day. This accelerated value, which was about 4-fold larger than the initial rate, is believed to be the direct consequence of amino acid deprivation since, in separate experiments, the increase was completely suppressed by the addition of plasma amino acids (Lardeux, B. R., and Mortimore, G. E. (1987) J. Biol. Chem. 262, 14514-14519). These findings demonstrate the potential value of cytidine as a marker for following moment-to-moment regulatory alterations in RNA degradation in the isolated liver or hepatocyte preparation.