Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes.
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).
Gross
M
M
Department of Pathology, University of Chicago, Illinois 60637.
Starn
T K
TK
Rundquist
C
C
Crow
P
P
White
J
J
Olin
A
A
Wagner
T
T
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000097:blood* / Q000302:isolation & purification; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D007700:Kinetics; D012343:RNA, Transfer / Q000302:isolation & purification / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000302:isolation & purification / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D013379:Substrate Specificity
D006882:Hydroxyapatites; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D012343:RNA, Transfer; D017886:Durapatite; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase