Inhibition of Ribonucleases by Ribonucleotides and Transition State Analogs in Cell-Free Extracts from Ehrlich Ascites Tumor Cells

Abstract

The ribonucleolytic breakdown of poly(U), poly(A), RNA transcribed from calf thymus DNA with Escherichia coli RNA polymerase, ribosomal RNA, tRNA and mengovirus RNA by an enzyme fraction obtained from a postribosomal supernatant of Ehrlich ascites tumor cells was investigated. The single-stranded homopolyribonucleotides were preferentially degraded by the enzyme fraction with the production of ribonucleoside 5''-monophosphates. The RNase activity was completely dependent on the presence of Mg2+ and was highest at Mg2+ and K+ concentrations optimal for cell-free protein synthesis. Ribonucleoside 5''-monophosphates, ribonucleoside 2''(3'')-monophosphates, ribonucleoside 2''(3''),5''-bisphosphates and transition state analogs consisting of vanadyl sulfate and either ribonucleosides or ribonucleoside 5''-monophosphates [1:1 M] inhibited the ribonucleolytic activity of the enzyme fraction. The ribonucleoside 2''(3''),5''-bisphosphates and the transition state analogs were the most effective inhibitors. However, only in the presence of ribonucleoside 2''(3''),5''-bisphosphates a concomitant stimulation by 50-60% of poly(U)-directed polyphenylalanine synthesis was observed; all the other RNase inhibitors tested also inhibited polypeptide synthesis. The results of preliminary experiments showed that poly(U) and ribonucleoside 2''(3''),5''-bisphosphates were well suited as ligands for affinity chromatography of ribonucleases from Ehrlich ascites tumor cells.