Rapid purification of tRNA(Lys) from rat liver.
Fast protein liquid chromatography (FPLC) system using Mono Q (HR 5/5) anion-exchange column chromatography followed by highly cross-linked urea-polyacrylamide gel electrophoresis (urea-PAGE) was used for the purification of lysine-specific tRNA (tRNA(Lys)) from rat liver. Crude tRNA from rat liver was fractionated with a linear gradient of NaCl (0.3-0.8 M) in triethanolamine-HCl buffer, pH 4.5, and the activity of tRNA(Lys) was found to elute between 0.51 and 0.57 M NaCl. Using this concentration range of NaCl, tRNA(Lys) was refractionated on the same column with a shallow gradient, where a single peak of tRNA(Lys) activity was obtained. tRNA(Lys)-rich fractions recovered from the second run were electrophoretically separated on 16% polyacrylamide-7 M urea gel into one major band and three minor bands. The major band showed a specific activity of 997 pmols/A260 U for tRNALys with a 43-fold purification and approximately 17% recovery. The minor bands displayed negligible or no activity for lysine. tRNA(Lys) obtained by this method was found to be homogeneous by competitive aminoacylation. The advantages of FPLC followed by urea-PAGE in the purification of an amino acid-specific tRNA over conventional column chromatography are discussed.
Kumar
A M
AM
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore.
Vulimiri
S V
SV
Nayak
R
R
D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification; D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry* / Q000201:enzymology; D008297:Male; D012357:RNA, Transfer, Lys / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012965:Sodium Chloride; D014508:Urea
D007202:Indicators and Reagents; D012357:RNA, Transfer, Lys; D012965:Sodium Chloride; D014508:Urea; D000604:Amino Acyl-tRNA Synthetases