A continuous spectrophotometric assay for the aminoacylation of transfer RNA by alanyl-transfer RNA synthetase.
A continuous spectrophotometric assay is described for the aminoacylation reaction catalyzed by Escherichia coli alanyl-transfer RNA synthetase. The assay is based on coupling the alanyl-tRNA synthetase-dependent formation of AMP to the lactate dehydrogenase oxidation of NADH. Oxidation of NADH, as monitored at 340 nm, is shown to be stoichiometric with the formation of alanyl-tRNA(Ala). This assay will facilitate the rapid accumulation and analysis of kinetic data for alanyl-tRNA synthetase and should be applicable to aminoacyl-tRNA synthetases in general.
Wu
M X
MX
Department of Biochemistry, Loma Linda University School of Medicine, California 92350.
Hill
K A
KA
D000215:Acylation; D000412:Alanine-tRNA Ligase / Q000378:metabolism*; D000586:Amination; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D012343:RNA, Transfer / Q000378:metabolism*; D011817:Rabbits; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods
D001426:Bacterial Proteins; D012343:RNA, Transfer; D000412:Alanine-tRNA Ligase