Development of a fluorescence polarization assay for peptidyl-tRNA hydrolase.
Peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Historically, the hydrolyzing activity of Pth has been assayed with radiolabeled N-blocked aminoacyl-tRNAs in assay systems that require the separation of radiolabeled amino acid from the N-blocked aminoacyl-tRNA complex. In the present study, we describe the development of a kinetic fluorescence polarization (FP) assay that enables measurements of Pth activity without the need to separate bound and free tracer. The hydrolyzing activity of Pth was determined by measuring the change in polarization values that resulted from the cleavage of a fluorescently labeled substrate (BODIPY-Lys-tRNA(Lys)). The data were analyzed using an equation describing first-order dissociation and the results showed that the experimental data correlated well with the theoretical curve. A runs test of the residuals showed that the experimental data did not significantly differ from the first-order model. The assay is adaptable to a multiwell format and is sensitive enough to detect Pth-like activity in bacterial cell lysate. The Pth FP assay provides a homogeneous and kinetic format for measuring Pth activity in vitro.
Bonin
Paul D
PD
Pharmacia Corporation, Kalamazoo, Michigan 4900, USA.
Erickson
Laurence A
LA
D000107:Acetylation; D001896:Boron Compounds; D002851:Chromatography, High Pressure Liquid; D019793:Fluorescein; D005454:Fluorescence Polarization / Q000379:methods*; D007700:Kinetics; D008250:Lysine-tRNA Ligase / Q000032:analysis* / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D008250:Lysine-tRNA Ligase; D019793:Fluorescein