Interaction between membrane functions and protein synthesis in reticulocytes. Isolation of RNase M, a membrane component inhibiting protein synthesis through specific endonucleolytic activity.
An inhibitor of protein synthesis has been isolated from reticulocyte membranes by solubilization with Triton X-100; it has been purified using heat treatment, filtration on Amicon filters, DEAE-cellulose ion-exchange chromatography and Sephadex G-75 gel chromatography. A final purification of 120-fold was achieved. The purified inhibitor was found to be 95% homogenous when run on a dodecylsulfate/polyacrylamide gel system. Three independent methods were used to estimate the molecular weight of the purified inhibitor: Sephadex G-75 gel chromatography, dodecylsulfate/polyacrylamide gel electrophoresis and sucrose gradient all confirmed that the purified inhibitor was a small molecule with a sedimentation coefficient of 0.7 S and a molecular weight ranging between 5000 and 8000. The purified inhibitor was shown to possess a specific endonucleolytic activity, degrading the 28-S species of ribosomal RNA to species sedimenting between 10 and 14 S. Due to its membrane localisation the name RNase M is proposed. The purified inhibitor's endonucleolytic activity was characterized with regard to its kinetics, concentration dependence, pH optimum and its requirements for divalent cations. Kinetics showed that RNase M retained its specificity after 60 min of incubation with the RNA substrate. Specificity was also demonstrated by incubating the polysomal RNA with high concentrations of purified enzyme. The pH optimum was found to be between pH 6 and pH 7, and the enzyme did not require divalent cations for its activity. Pancreatic RNase B used at a similar protein synthesis inhibitory concentration as the RNase M caused a complete breakdown of ribosomal RNA to oligonucleotides and mononucleotides. The possible biological significance of the purified inhibitor in regulating protein synthesis in the maturing reticulocyte is discussed.
D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis*; D004720:Endonucleases / Q000097:blood / Q000302:isolation & purification; D004722:Endoribonucleases; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012260:Ribonucleases / Q000097:blood* / Q000302:isolation & purification; D012270:Ribosomes / Q000378:metabolism
D001798:Blood Proteins; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; C022538:ribonuclease M