Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis.
Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. 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 microM 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 due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S 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 [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.
Morishita
R
R
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan.
Kawagoshi
A
A
Sawasaki
T
T
Madin
K
K
Ogasawara
T
T
Oka
T
T
Endo
Y
Y
D000818:Animals; D002474:Cell-Free System; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D012260:Ribonucleases / Q000378:metabolism*; D013379:Substrate Specificity
D006360:Heat-Shock Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C097211:Rida protein, rat; D012260:Ribonucleases