Studies on Transfer Ribonucleic Acid-Ribosome Complexes

The reaction of puromycin with nascent peptides on polyribosomes was used to study transpeptidation on mammalian polysomes. This reaction on polyribosomes should be more nearly a model of transpeptidation in the intact cell than studies of peptide bond synthesis with synthetic donors and washed ribosomes. With the use of this assay for transpeptidation, it was possible to show that a single K+ or Na+ is necessary for transpeptidation; that protonation of a single group with a pKa equal to about 7.7 inactivates the transpeptidase complex; that with respect to protonation of this group H+ is a noncompetitive inhibitor of peptidyl-puromycin synthesis; that protonation of a single moiety with a pKa of 9.4 activates the transpeptidase complex; that Mn++ can substitute for Mg++, but that Ca++, spermidine, spermine, and putrescine do so only very poorly; and that the Km for puromycin in this assay is about 8 x 10-6 m. In addition, the effect of pH on transpeptidation is analyzed to directly yield the pKa and the number of ionizable groups involved in the rate-limiting reaction by a simplified method which may have more general applicability.