Abstract
We have identified an activity in rabbit reticulocyte lysate as peptidyl-tRNA hydrolase, based upon its ability to hydrolyze native reticulocyte peptidyl-tRNA, isolated from polyribosomes, and N-acylaminoacyl-tRNA, and its inability to hydrolyze aminoacyl-tRNA, precisely the same substrate specificity previously reported for peptidyl-tRNA hydrolase from bacteria or yeast. The physiological role of the reticulocyte enzyme may be to hydrolyze and recycle peptidyl-tRNA that has dissociated prematurely from elongating ribosomes, as suggested for the bacterial and yeast enzymes, since reticulocyte peptidyl-tRNA hydrolase is completely incapable of hydrolyzing peptidyl-tRNA that is still bound to polyribosomes. We have purified reticulocyte peptidyl-tRNA hydrolase over 5,000-fold from the postribosomal supernatant with a yield of 14%. The purified product shows a 72-kDa band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis that has co-purified with enzyme activity and comprises about 90% of the total stained protein, strongly suggesting that the 72-kDa protein is the enzyme. Sucrose density gradient analysis indicates an apparent molecular mass for the native enzyme of 65 kDa, implying that it is a single polypeptide chain. The enzyme is almost completely inactive in the absence of a divalent cation: Mg2+ (1-2 mM) promotes activity best, Mn2+ is partly effective, and Ca2+ and spermidine are ineffective. The hydrolase shows a Km of 0.60 microM and Vmax of 7.1 nmol/min/mg with reticulocyte peptidyl-tRNA, a Km of 60 nM and Vmax of 14 nmol/min/mg with Escherichia coli fMet-tRNA(fMet), and a Km of 100 nM and Vmax of 2.2 nmol/min/mg with yeast N-acetyl-Phe-tRNA(Phe). The enzyme has a pH optimum of 7.0-7.25, it is inactivated by heat (60 degrees C for 5 min), and its activity is almost completely inhibited by pretreatment with N-ethylmaleimide or incubation with 20 mM phosphate. The fact that the enzyme hydrolyzes E. coli but not yeast or reticulocyte fMet-tRNA(fMet) may be explained, at least in part, by structural similarities between prokaryotic tRNA(fMet) and eukaryotic elongator tRNA that are not shared by eukaryotic tRNA(fMet).
Highlights
We have identifiedan activity in rabbit reticulocyte tidyl-tRNA and N-acylaminoacyl-tRNA but not aminoacyllysate as peptidyl-tRNA hydrolase, basedupon its abil- tRNA, has been purified and characterized from bacteria
In recentstudies relating thestructure of different initiatortRNAs to their function when added to rabbit reticulocyte lysate, we found, unexpectedly, that there is rapid enzymatic hydrolysis of bacterial M e t tRNAyt (9).Purification andcharacterization of this enzyme from rabbit reticulocyte lysate, reported here, indicate that it reticulocyte peptidyl-tRNA hydrolase over 6,000-fold is a peptidyl-tRNA hydrolase, since, like the bacterial and from the postribosomal supernatant with a yield of yeast enzymes, it requires a divalent cation for activity and
NM and VmnXof 2.2 nmol/min/mg with yeast N-acetylPhe-tRNAPhe.The enzyme has a pH optimum of 7.07.25, it is inactivated by hea(t60 OC for 5 min), and its ation (Step 3) have been described previously (12). this fraction (Step 3) was used for further purification, one should note that about 10% of the total peptidyl-tRNA hydrolase in the reticulocyte lysate is found in the0.50M KC1 wash of the ribosomes,about one-third of the enzyme activity in the postribosomal supernatant remains soluble at pH5.2,and about one-fourth of the enzymeactivity in the pH5.2precipitate remains solublein 40% saturated ammonium sulfate
Summary
We have identifiedan activity in rabbit reticulocyte tidyl-tRNA and N-acylaminoacyl-tRNA but not aminoacyllysate as peptidyl-tRNA hydrolase, basedupon its abil- tRNA, has been purified and characterized from bacteria TRNA hydrolase, which elutes a t about 0.05 to 0.11 M potassium Unlabeled peptidyl-tRNA was prepared from resuspended rabbit phosphate, was pooled and concentrated by ultrafiltration to give reticulocyte ribosomes that had been previously extracted with 0.5 M
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