ABSTRACT
Monospecific polyclonal antibodies (pAbs) against highly purified bovine seryl-tRNA synthetase (SerRS, EC 6.1.1.1) were prepared and their specificity tested. The interactions of pAbs with SerRS from different organisms were investigated by protein immunoblotting and ELISA methods. pAbs inhibit eukaryotic SerRS aminoacylating activity and exert no effect on SerRS activity from prokaryotes. It is proposed that prokaryotic and eukaryotic SerRS evolve from different ancestor genes.
Subject(s)
Serine-tRNA Ligase/metabolism , Animals , Antibodies , Blotting, Western , Cattle , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Escherichia coli/enzymology , Immunohistochemistry , Liver/enzymology , Rabbits , Serine-tRNA Ligase/immunologyABSTRACT
The interaction of the cow mammary gland tRNA(IAGLeu), having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by the alkylation with ethylnitrosourea. It was shown that leucyl-tRNA synthetase protects from alkylation 3'-phosphates of the nucleotides 12-13 in D-loop, 23-24 in D-stem and 37-43 in the anticodon arm of tRNA(IAGLeu). All regions of interaction with the aminoacyl-tRNA synthetase are located in the same plane of tRNA whereas the long variable loop is in another plane.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Mammary Glands, Animal/metabolism , RNA, Transfer, Leu/genetics , Alkylation , Animals , Autoradiography , Base Sequence , Cattle , Electrophoresis, Polyacrylamide Gel , Female , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Transfer, Leu/metabolismABSTRACT
The structural accessibility of tryptophan residues in leucyl-tRNA synthetase from cow mammary gland has been studied using chemical modifications by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. The modifications were monitored by UV absorbance and intrinsic fluorescence of the enzyme's tryptophan residues. Under native conditions, at pH 7,8, only two exposed tryptophan residues are modified in each subunit of the dimeric enzyme. Under denaturing conditions, in 6 M guanidine hydrochloride solution, internal tryptophan residues are also modified as a consequence of unfolding of the native tertiary structure of the enzyme. Modifications of tryptophan residues resulted in inactivation of leucyl-tRNA synthetase both in aminoacylation and ATP-PPi exchange reactions. In the specific complex of leucyl-tRNA synthetase with the cognate tRNALeu one of exposed tryptophan residues is protected by tRNALeu and is not modified by the above reagents.
Subject(s)
2-Hydroxy-5-nitrobenzyl Bromide , Amino Acyl-tRNA Synthetases , Bromosuccinimide , Leucine-tRNA Ligase , Nitrophenols , Succinimides , Tryptophan/analysis , Amino Acyl-tRNA Synthetases/metabolism , Animals , Cattle , Chemical Phenomena , Chemistry , Female , Fluorescence , In Vitro Techniques , Indicators and Reagents , Leucine-tRNA Ligase/metabolism , Mammary Glands, Animal/enzymology , Protein Denaturation , Spectrophotometry, UltravioletABSTRACT
Three forms (E1, E2 and E3) of leucyl-tRNA synthetase (LeuRS) were separated by DEAE-cellulose chromatography of total aminoacyl-tRNA synthetases from cow lactating mammary gland. The method of purification of all three components is described. E1 is a dimeric molecule (alpha 2) of molecular weight 182 000. Two other forms of molecular weight 67 000 and 64,000 consist of a single polypeptide chain as determined by polyacrylamide gel electrophoresis. Optimum conditions and kinetic parameters of leucyl-tRNA formation were studied for every enzyme form. The low values of Vmax and thermostability are characteristic of E3. All forms of LeuRS interact with 6 isoaccepting tRNA(Leu) from lactating mammary gland and can activate leucine in the absence of tRNA. E2 and E3 are supposed to derive from the native enzyme by endogenous proteolysis. The physico-chemical properties of native LeuRS from lactating mammary gland are compared with those of LeuRS's from other sources.
