Purification and Characterization of an Inhibitor of Elongation Factor G-dependent Guanosine Triphosphatase Reaction of Ribosomes from Ribosome Wash of Escherichia coli Q13

Abstract

Abstract An inhibitor of the elongation factor G-dependent uncoupled GTPase reaction of ribosomes has been purified from a solution obtained by washing ribosomes of Escherichia coli Q13 with a buffer containing 0.5 m NH4Cl. The molecular weight of the inhibitor as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration was 23,000 and 19,000, respectively. The inhibitor was specific for the elongation factor G-dependent uncoupled GTPase reaction of 70 S ribosomal particles and did not inhibit the elongation factor G-dependent GTPase reaction of 50 S ribosomal subunit particles. Kinetic studies on the inhibition of the GTPase reaction indicated that the inhibitor was competitive with ribosomes and noncompetitive with elongation factor G and GTP in the GTPase reaction. The ribosomes, which were incubated with the inhibitor and then separated from the free inhibitor, had lower GTPase activity than the ribosomes incubated without the inhibitor. These results are taken as evidence that the inhibitor acts on ribosomes and inhibits the elongation factor G-dependent uncoupled GTPase reaction of ribosomes.