Physiological studies of evolutionaly conserved-perchloric acid soluble-protein (PSP) from rat liver

大鼠利伯全氯酸溶解蛋白（L-PSP）是无细胞蛋白合成的潜在抑制剂;不管怎样，它的作用机制还不为人所知。在这里，我们展示蛋白质是一种独特的核糖核酸酶，并且这种活动对翻译的抑制是负责任的。将全氯酸溶解蛋白添加到一个兔子视网膜无细胞系统中的6.2 μM LED集中在一个几乎完全抑制蛋白质合成的方法中。动力学并不像那些受海文控制的抑制剂，它们是在启动阶段发生的蛋白质。这一现象似乎是全氯酸易失蛋白的内源性核素活性，因为L-PSP直接影响mRNA模板活性和诱导体对视网膜酸多态性进入80 S核糖体的排斥，甚至在环氯类化合物的存在中。这些效果与真实性一样好，与L-PSP重新组合。通过分析[α -Iye D132 Iye D1 P] UTF标记的mRNA在蛋白显示出ribonucleotide 3'-monophosphates Ap、Gp、UP和Gp中培养，直接证据表明蛋白质是一种endribonuclease。当5'-或3'-32 P标记的5S rRNA是一个基底， L-PSP清除的磷酸酶键仅在分子区域的单个链中。

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not unknown. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 μM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [α -ィイD132ィエD1P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleotide 3'-monophosphates Ap, Gp, UP and Gp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single strand region of the molecule.