Purification of the yeast mitochondrial methionyl-tRNA synthetase. Common and distinctive features of the cytoplasmic and mitochondrial isoenzymes.
Yeast-mitochondrial methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNA(Met)-Sepharose, Agarose-hexyl-AMP) to yield to a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl-tRNA synthetase (Mr 85,000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65,000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152,000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA(Met) are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNA(Met). Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes. Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.
Schwob
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Institut de Biologie Moléculaire et Cellulaire du CNRS, Laboratoire de Biochemie, Strasbourg, France.
Sanni
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Fasiolo
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Martin
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RP
D000215:Acylation; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D000906:Antibodies / Q000494:pharmacology; D000917:Antibody Formation; D001665:Binding Sites / Q000187:drug effects; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin; D007527:Isoenzymes / Q000302:isolation & purification; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000906:Antibodies; D007527:Isoenzymes; D006493:Heparin; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase