Isolation of prolyl-tRNA synthetase as a free form and as a form associated with glutamyl-tRNA synthetase.
Rat liver prolyl-tRNA synthetase was purified as a dimer of M(r) 60,000 subunits not associated with other aminoacyl-tRNA synthetases and as a form associated with glutamyl-tRNA synthetase. Proteolysis of the dimeric enzyme generated a less active form with M(r) 52,000 subunits and an inactive form with M(r) 40,000 subunits. A second species was isolated with polypeptides of M(r) 60,000 and 150,000. This form dissociated during gel filtration chromatography being partially resolved into the M(r) 150,000 and 60,000 components; glutamyl-tRNA synthetase was associated with the larger polypeptide and prolyl-tRNA synthetase with the smaller component. Antibodies against the M(r) 60,000 polypeptide reacted with the M(r) 60,000 and 150,000 polypeptides. Gel filtration of extracts revealed multiple forms of prolyl- and glutamyl-tRNA synthetase. Antibody against the M(r) 60,000 component detected the M(r) 60,000 and 150,000 polypeptides throughout the chromatogram; these forms could be partially separated by polyethylene glycol fractionation. The M(r) 150,000 and 60,000 polypeptides were detected by Western blot analysis of crude extracts prepared under several conditions. Antibody to prolyl-tRNA synthetase reacted with a M(r) 150,000 polypeptide of the aminoacyl-tRNA synthetase core complex identified previously as glutamyl-tRNA synthetase.
Ting
S M
SM
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699-0008.
Bogner
P
P
Dignam
J D
JD
D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005975:Glutamate-tRNA Ligase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D051381:Rats
D000596:Amino Acids; D046911:Macromolecular Substances; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase; D005975:Glutamate-tRNA Ligase