Presence of role of the 5SrRNA-L5 protein complex (5SRNP) in the threonyl- and histidyl-tRNA synthetase complex in rat liver cytosol.
A complex containing Thr-RS and His-RS was purified about 1000 to 2000-fold from rat liver cytosol by successive column chromatographies on Sephadex G-200, Phenyl-Sepharose CL-4B, and tRNA-Sepharose. The ratio of the specific activity of Thr-RS and His-RS was relatively constant throughout the purification steps, suggesting that the two synthetases were co-purified as a complex. Chromatographic analyses of the tRNA-Sepharose fraction by Sephadex G-150 column chromatography showed the presence of a hybrid form of the Thr-RS monomer and the His-RS monomer in addition to dimer forms of both enzymes from the pattern of activity of both enzymes. The monomer form of Thr-RS showed high activity comparable to the dimer form and the monomer form of His-RS showed definite activity. An association form of Thr-RS and His-RS dimers was detected by Sephadex G-200 chromatography of rat liver cytosol. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 55SrRNA blot analysis of the tRNA-Sepharose fraction using an antibody against ribosomal protein L5, showed the presence of ribosomal protein L5 in this fraction. These findings suggest that the presence of a 5SRNA-L5 protein complex (5SRNP) in the Thr-RS and His-RS complex. 5SRNP enhanced the activity of Thr-RS in a freshly prepared tRNA-Sepharose fraction. It also enhanced the activity of the rat liver cytosol for the attachment of [3H]threonine to endogenous tRNA. This activity was inhibited by an antibody against protein L5, and the inhibition was reversed by addition of 5SRNP. These results indicate that 5SRNP plays a role as a positive effector of Thr-RS in the complex.
Ogata
K
K
Institute for Gene Expression, Dobashi Kyoritsu Hospital, Ehime, Japan.
Kurahashi
A
A
Nishiyama
C
C
Terao
K
K
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006643:Histidine-tRNA Ligase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D012341:RNA, Ribosomal, 5S / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D012269:Ribosomal Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D013914:Threonine-tRNA Ligase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D012341:RNA, Ribosomal, 5S; D012269:Ribosomal Proteins; C029742:ribosomal protein L5; D006643:Histidine-tRNA Ligase; D013914:Threonine-tRNA Ligase