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

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free proteinsynthesis;however,它的行为机制不为人知，我们在这里展示的证明是一个独特的权利和保障this activity is responsible for the inhibition of translation. the addition of perchloricacid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 μM led to蛋白质synthesis almost complete inhibition of protein synthesis The kinetics are unlike those ofhemin - controlled inhibitor,a protein that acts at the initiation step. the inhibition appears to be an endoribonucleolyticactivity of perchloric acid-soluble protein because - psp直接affects mRNA template activityand induces disaggregation of the reticulocyte polysomes into 80s ribosomes，即使在cycloheximide presence of cycloheximide.这些effects被监测与authentic as well asrecombinant - psp . Analysis by think -layer chromatography of [α - d - 132 - D1P]UTP-labeled mRNAincubated 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 -labeled5s rRNA was the substratepsp cleaved phosphodiester bonds only in the single strand region of the molecule。

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.