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

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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.