Purification and properties of alanine tRNA synthetase from Escherichia coli A tetramer of identical subunits.
Escherichia coli alanine tRNA synthetase has been purified from a strain which carries the gene on a recombinant pBR322 plasmid. Several per cent of the soluble protein can be obtained as alanine tRNA synthetase in the plasmid containing host cell. The enzyme was proven to be an alpha 4 tetramer with a Mr = 380,000 by the following criteria: i) a single band of Mr = 95,000 was found in sodium dodecyl sulfate gel electrophoresis; ii) gel filtration chromatography gives a single peak at a Mr = 360,000 for the native enzyme; iii) dimethyl suberimidate cross-linking indicates a tetrameric structure; iv) a single undecapeptide N-terminal sequence was found which is Ser-Lys-Ser-Thr-Ala-Glu-Ile-Arg-Gln-Ala-Phe. Limited proteolysis generates a fragment of the native enzyme. The fragment has a Mr = 48,000 (one half that of the native subunit) and, in contrast to the native enzyme, oligomerization of the fragment could be not detected either by gel filtration chromatography or by dimethyl suberimidate cross-linking. The fragment is derived from the NH2-terminal half of the native subunit as shown by their common decapeptide NH2-terminal amino acid sequences. The ATP-PPi exchange activity of 1 mol of fragment is identical with that of 1 mol of native subunit, but aminoacylation activity is absent from the fragment. Therefore, in the fragment, the aminoacyl adenylate formation activity is cleanly separated from tRNA aminoacylation and subunit association properties. These results also mean that, with respect to aminoacyl adenylate formation activity, each subunit in the native tetramer acts independently.
D000412:Alanine-tRNA Ligase / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D004120:Dimethyl Suberimidate; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight
D000596:Amino Acids; D046911:Macromolecular Substances; D004120:Dimethyl Suberimidate; D000604:Amino Acyl-tRNA Synthetases; D000412:Alanine-tRNA Ligase