Do yeast aminoacyl-tRNA synthetases exist as soluble enzymes within the cytoplasm?

The aminoacyl-tRNA synthetases from a crude extract of yeast were shown to bind to heparin-Ultrogel through ionic interactions, in conditions where the corresponding enzymes from Escherichia coli did not. The behaviour of purified lysyl-tRNA synthetases from yeast and E. coli was examined in detail. The native dimeric enzyme from yeast (Mr 2 X 73000) strongly interacted with immobilized heparin or tRNA, as well as with negatively charged liposomes, in conditions where the corresponding native enzyme from E. coli (Mr 2 X 65000) displayed no affinity for these supports. Moreover, the aptitude of the native enzyme from yeast to interact with polyanionic carriers was lost on proteolytic conversion to a fully active modified dimer of Mr 2 X 65500. A structural model is proposed, according to which each subunit of yeast lysyl-tRNA synthetase is composed of a functional domain similar in size to that of the prokaryotic enzyme, contiguous to a 'binding' domain responsible for association to negatively charged carriers. The evolutionary acquisition of this property by lower eukaryotic aminoacyl-tRNA synthetases suggests that it fulfils an important function in vivo, unrelated to catalysis. We propose that it promotes the compartmentalization of these enzymes within the cytoplasm, through associations with as yet unidentified, negatively charged components, by electrostatic interactions too fragile to withstand the usual extraction conditions.     

A complex from cultured Chinese hamster ovary cells containing nine aminoacyl-tRNA synthetases. Thermolabile leucyl-tRNA synthetase from the tsH1 mutant cell line is an integral component of this complex

The size distribution of the 20 aminoacyl-tRNA synthetases from wild-type Chinese hamster ovary (CHO) cells and from the mutant cell line tsH1, containing a temperature-sensitive leucyl-tRNA synthetase, was determined by gel filtration. Nine aminoacyl-tRNA synthetases, specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine and proline, which coeluted as high-Mr entities (Mr approximately 1.2 X 10(6)), were further co-purified to yield a multienzyme complex, the polypeptide composition of which was identical to that previously determined for the complex from rabbit liver. Immunoprecipitates obtained from crude extracts of wild-type and tsH1 mutant cells, using specific antibodies directed to the lysyl-tRNA or methionyl-tRNA synthetase components of the complex, displayed the same polypeptide compositions as that of the purified complex, thereby establishing the heterotypic nature of this complex. Although the activity of leucyl-tRNA synthetase from the mutant cells, grown at a permissive temperature, was low compared to that from the wild-type, the polypeptide of Mr 129 000, corresponding to this enzyme, was present in similar amounts and occurred exclusively as a component of the high-Mr complex. Finally, we report that attempts to demonstrate phosphorylation of the components of the complex from cultured CHO, HeLa and C3 cells were unsuccessful.     

The C-terminal appended domain of human cytosolic leucyl-tRNA synthetase is indispensable in its interaction with arginyl-tRNA synthetase in the multi-tRNA synthetase complex

Human cytosolic leucyl-tRNA synthetase is one component of a macromolecular aminoacyl-tRNA synthetase complex. This is unlike prokaryotic and lower eukaryotic LeuRSs that exist as free soluble enzymes. There is little known about it, since the purified enzyme has been unavailable. Herein, human cytosolic leucyl-tRNA synthetase was heterologously expressed in a baculovirus system and purified to homogeneity. The molecular mass (135 kDa) of the enzyme is close to the theoretical value derived from its cDNA. The kinetic constants of the enzyme for ATP, leucine, and tRNA(Leu) in the ATP-PP(i) exchange and tRNA leucylation reactions were determined, and the results showed that it is quite active as a free enzyme. Human cytosolic leucyl-tRNA synthetase expressed in human 293 T cells localizes predominantly to the cytosol. Additionally, it is found to have a long C-terminal extension that is absent from bacterial and yeast LeuRSs. A C-terminal 89-amino acid truncated human cytosolic leucyl-tRNA synthetase was constructed and purified, and the catalytic activities, thermal stability, and subcellular location were found to be almost identical to native enzyme. In vivo and in vitro experiments, however, show that the C-terminal extension of human cytosolic leucyl-tRNA synthetase is indispensable for its interaction with the N-terminal of human cytosolic arginyl-tRNA synthetase in the macromolecular complex. Our results also indicate that the two molecules interact with each other only through their appended domains.     

Phaseolus vulgaris cytoplasmic leucyl-tRNA synthetase. Purification and comparison of its catalytic, structural, and immunological properties with those of the chloroplastic enzyme

The cytoplasmic leucyl-tRNA synthetase was purified from bean (Phaseolus vulgaris) leaves. After ammonium sulfate fractionation and chromatography on Sephadex G-50, DEAE-cellulose, hydroxylapatite, and phosphocellulose, complete purification was achieved by blue Sepharose CL-6B chromatography using specific elution with pure yeast tRNALeu1. The enzyme was purified 1050-fold and had a specific activity of 940 nmol of leucyl-tRNA formed/min/mg of protein. Polyacrylamide gel electrophoresis of the native enzyme showed one band, but the denatured enzyme showed two bands. These two protein bands are structurally related. The smallest protein appears to be a cleavage product from the largest one, suggesting the presence of a sensitive cleavage site in the cytoplasmic leucyl-tRNA synthetase. The cytoplasmic enzyme is a monomer (Mr = 130,000), larger than its chloroplastic counterpart (Mr = 120,000). The two enzymes differ in their substrate (tRNA) specificity, tryptic peptide map, and amino acid composition. Antibodies were raised against the cytoplasmic enzyme and against the chloroplastic enzyme and no cross-immunological reaction was detected, showing that the two enzymes do not share any antigenic determinant. Taken together, these results suggest that P. vulgaris cytoplasmic and chloroplastic leucyl-tRNA synthetases are coded for by different genes.     

Valyl-tRNA synthetase from rabbit liver. II. The enzyme derived from the high-Mr complex displays hydrophobic as well as polyanion-binding properties

The preceding paper (Bec, G., Kerjan, P., Zha, X.D., and Waller, J.P. (1989) J. Biol. Chem. 264, 21131-21137) described the purification to apparent homogeneity from rabbit liver, of a heterotypic complex comprising valyl-tRNA synthetase and Elongation Factor 1H. In the present study, valyl-tRNA synthetase was dissociated and separated from the other components of this complex by hydroxylapatite chromatography in the presence of 0.5 M NaSCN. The properties of the homogeneous mammalian enzyme were compared to those of the corresponding enzyme from yeast. Both behaved as monomeric entities, with apparent molecular masses of 140 and 125 kDa, respectively. Furthermore, both displayed strong affinity toward the polyanionic support heparin-Ultrogel, a property not manifested by the corresponding prokaryotic enzyme. However, unlike the yeast enzyme, that of mammalian origin additionally exhibited hydrophobic properties, as reflected by its affinity toward phenyl-Sepharose. A structural model is proposed according to which mammalian valyl-tRNA synthetase has conserved the polycationic N-terminal domain that distinguishes the corresponding lower eukaryotic enzyme from its prokaryotic counterpart, while acquiring a hydrophobic domain most likely responsible for its association to Elongation Factor 1H.     

Histidyl-tRNA synthetase, the myositis Jo-1 antigen, is cytoplasmic and unassociated with the cytoskeletal framework

The myositis-specific anti-Jo-1 autoantibody, which is directed against histidyl-tRNA-synthetase, is found in 30% of polymyositis patients. The Jo-1 antigen has been reported to be a nuclear antigen by some authors. On the contrary we show that less than 2% of the total histidyl-tRNA and lysyl-tRNA synthetase activities are associated with purified rat liver nuclei or the hepatocyte intermediate filament-nuclear fraction. In the presence of polyethylene glycol, in which the high Mr multi-enzyme complex containing lysyl-tRNA synthetase is insoluble, 65% of the lysyl-tRNA synthetase and only 15% of histidyl-tRNA synthetase activities remained associated with the cytoskeletal framework. The Jo-1 antigen exhibited a diffuse granular cytoplasmic distribution in cultured rat hepatocytes as determined by indirect immunofluorescent microscopy. Hence, the Jo-1 antigen is cytoplasmic and unassociated with the cytoskeletal framework or high Mr synthetase complex in situ.     

Rapid isolation of lysyl-tRNA synthetase from rat liver

The high molecular weight aminoacyl-tRNA synthetase complex (the 24S complex) was isolated from rat liver by ultracentrifugation. The lysyl-tRNA synthetase (E.C. 6.1.1.6) was selectively dissociated by hydrophobic interaction chromatography on 1,6 diaminohexyl agarose followed by hydroxylapatite chromatography and DEAE chromatography. The lysyl-tRNA synthetase dissociated from the 24S synthetase complex was purified approximately to 2700 fold with 14% yield.     

Alteration of aminoacyl-tRNA synthetase activities by phosphorylation with casein kinase I

The phosphorylation of a highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes by the cyclic nucleotide-independent protein kinase, casein kinase I, has been examined, and the effects of phosphorylation on the synthetase activities were determined. The synthetase complex, purified as described (Kellermann, O., Tonetti, H., Brevet, A., Mirande, M., Pailliez, J.-P., and Waller, J.-P. (1982) J. Biol. Chem. 257, 11041-11048), contains seven aminoacyl-tRNA synthetases and four unidentified proteins and is free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP results in the phosphorylation of four synthetases, namely, glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I alters binding of the aminoacyl-tRNA synthetase complex to tRNA-Sepharose. The phosphorylated synthetase complex elutes from tRNA-Sepharose at 190 mM NaCl, while the nonphosphorylated complex elutes at 275 mM NaCl. Phosphorylation by casein kinase I results in a significant inhibition of aminoacylation by the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases; the activities of the nonphosphorylated synthetases remain unchanged. These data indicate that phosphorylation of aminoacyl-tRNA synthetases in the high molecular weight complex alters the activities of these enzymes. One of the unidentified proteins present in the complex (Mr 37,000) is also highly phosphorylated by casein kinase I. From a comparison of the properties and phosphopeptide pattern of this protein with that of casein kinase I, it appears that the Mr 37,000 protein in the synthetase complex is an inactive form of casein kinase I. This observation provides further evidence for a physiological role for casein kinase I in regulating synthetase activities.     

Purification and characterization of the isoleucyl-tRNA synthetase component from the high molecular weight complex of sheep liver: a hydrophobic metalloprotein

Native isoleucyl-tRNA synthetase and a structurally modified form of methionyl-tRNA synthetase were purified to homogeneity following trypsinolysis of the high molecular weight complex from sheep liver containing eight aminoacyl-tRNA synthetases. The correspondence between purified isoleucyl-tRNA synthetase and the previously unassigned polypeptide component of Mr 139 000 was established. It is shown that dissociation of this enzyme from the complex has no discernible effect on its kinetic parameters. Both isoleucyl- and methionyl-tRNA synthetases contain one zinc ion per polypeptide chain. In both cases, removal of the metal ion by chelating agents leads to an inactive apoenzyme. As the trypsin-modified methionyl-tRNA synthetase has lost the ability to associate with other components of the complex [Mirande, M., Kellermann, O., & Waller, J. P. (1982) J. Biol. Chem. 257, 11049-11055], the zinc ion is unlikely to be involved in complex formation. While native purified isoleucyl-tRNA synthetase displays hydrophobic properties, trypsin-modified methionyl-tRNA synthetase does not. It is suggested that the assembly of the amino-acyl-tRNA synthetase complex is mediated by hydrophobic domains present in these enzymes.     

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.     

Comparison of the enzymatic behavior of high molecular weight and free lysyl-tRNA synthetase from rat liver: kinetic analysis of lysylation of tRNA

Lysyl-tRNA synthetase occurs in the high molecular weight form in rat liver. The high molecular weight lysyl-tRNA synthetase has been previously demonstrated to exist as multienzyme complexes of aminoacyl-tRNA synthetases. The multienzyme complexes can be dissociated by hydrophobic interaction chromatography and yield fully active, free lysyl-tRNA synthetase. The free form is found to be twice as active as the complexed form in lysylation. Bisubstrate and product inhibition kinetics of lysylation are systematically carried out for highly purified free lysyl-tRNA synthetase and the 18 S synthetase complex. Surprisingly, the two enzyme forms exhibit distinctly different kinetic patterns in bisubstrate and product inhibition kinetics under identical conditions. The 18 S synthetase complex shows kinetic patterns consistent with an ordered bi uni uni bi ping pong mechanism, while the results of free lysyl-tRNA synthetase do not. We conclude that structural organization of lysyl-tRNA synthetase beyond quaternary structure of proteins may alter the enzyme behavior.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.     

Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5. Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.     

Engineering mammalian aspartyl-tRNA synthetase to probe structural features mediating its association with the multisynthetase complex.

Aspartyl-tRNA synthetase from higher eukaryotes is a component of a multienzyme complex comprising nine aminoacyl-tRNA synthetases. The cDNA encoding cytoplasmic rat liver aspartyl-tRNA synthetase was previously cloned and sequenced. This work reports the identification of structural features responsible for its association within the multisynthetase complex. Mutant and chimeric proteins have been expressed in mammalian cells and their structural behavior analyzed. A wild-type rat liver aspartyl-tRNA synthetase, expressed in Chinese hamster ovary (CHO) cells, associates within the complex from CHO cells, whereas a mutant enzyme with a deletion of 34 amino acids from its amino-terminal extremity does not. A chimeric enzyme, made of the amino-terminal moiety of rat liver aspartyl-tRNA synthetase fused to the catalytic domain of yeast lysyl-tRNA synthetase, has been expressed in Lys-101 cells, a CHO cell line with a temperature-sensitive lysyl-tRNA synthetase. The fusion protein is stable in vivo, does not associate within the multisynthetase complex and cannot restore normal growth of the mutant cells. These results establish that the 3.7-kDa amino-terminal moiety of mammalian aspartyl-tRNA synthetase mediates its association with the other components of the complex. In addition, the finding that yeast lysyl-tRNA synthetase cannot replace the aspartyl-tRNA synthetase component of the mammalian complex, indicates that interactions between neighbouring enzymes also play a prominent role in stabilization of this multienzyme structure and strengthened the view that the multisynthetase complex is a discrete entity with a well-defined structural organization.     

Glycyl- and alanyl-tRNA synthetases from Bombyx mori. Purification and properties

Glycyl- and alanyl-tRNA synthetases have been purified from an extract of Bombyx mori posterior silk glands by a procedure that allows for the simultaneous isolation of both enzymes. Glycyl-tRNA synthetase is a dimer of Mr = 160,000 consisting of similar or identical subunits, whereas alanyl-tRNA synthetase is a monomer of Mr = 110,000 to 115,000. The abundance of these two enzymes in the posterior silk gland is consistent with the observed adaptation of this organ to the production of the silk protein, fibroin. The two enzymes are similar in oligomeric structure to the corresponding enzymes in Saccharomyces cerevisiae, but dissimilar from their counterparts in Escherichia coli.     

Isolation, characterization, and structural organization of 10-formyltetrahydrofolate synthetase from spinach leaves

One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes. In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide. The structural organization of these enzymes in plants had not previously been examined. We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it. The protein was a dimer with a subunit molecular weight of Mr = 67,000. The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively. The enzyme required both monovalent and divalent cations for maximum activity. The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column. On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted. In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves. These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional. Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.     

Renaturation of rabbit liver aminoacyl-tRNA synthetases by 80S ribosomes.

Protein biosynthesis machinery is thought to be mostly compartmentalised within the mammalian cell, involving direct interactions between different components of the translation apparatus. The present research concerns the functional meaning of the interaction between the rabbit liver aminoacyl-tRNA synthetases and 80S ribosomes. We have shown that rabbit liver 80S ribosomes are able to enhance the activity of leucyl-tRNA synthetase, which is a component of high-molecular weight aminoacyl-tRNA synthetase complex, and phenylalanyl-tRNA synthetase not associated within this complex. The ribosomes increase the initial rate of both the total reaction of tRNA aminoacylation and the first step of this reaction, the formation of leucyladenylate. Moreover, a positive cooperativity of the tRNA interaction with two binding sites of leucyl-tRNA synthetase is also increased in the presence of highly purified 80S ribosomes. The effect of 80S ribosomes on partly denatured leucyl-tRNA synthetase and phenylalanyl-tRNA synthetase and the protection by 80S ribosomes of both enzymes against inactivation indicate a refolding and stabilising capacity of the ribosomes. It is concluded that the interaction of aminoacyl-tRNA synthetases and 80S ribosomes is important for the maintenance of an active conformation of the enzymes.     

The NH2-terminal extension of rat liver arginyl-tRNA synthetase is responsible for its hydrophobic properties

Rat liver arginyl-tRNA synthetase is found in extracts either as a component (Mr = 72,000) of the multienzyme aminoacyl-tRNA synthetase complex or as a low molecular weight (Mr = 60,000) free protein. The two forms are thought to be identical except for an extra peptide extension at the NH2-terminus of the larger form which is required for its association with the complex, but is unessential for catalytic activity. It has been suggested that interactions among synthetases in the multienzyme complex are mediated by hydrophobic domains on these peptide extensions of the individual proteins. To test this model we have purified to homogeneity the larger form of arginyl-tRNA synthetase and compared its hydrophobicity to that of its low molecular weight counterpart. We show that whereas the smaller protein displays no hydrophobic character, the larger protein demonstrates a high degree of hydrophobicity. No lipid modification was found on the high molecular weight protein indicating that the amino acid sequence itself is responsible for its hydrophobic properties. These findings support the proposed model for synthetase association within the multienzyme complex.     

Methionyl-tRNA synthetase from sheep liver. Purification of a fully active monomer derived from high-molecular-weight complexes by trypsin treatment. Evidence for immunological cross-reaction with the corresponding enzyme from sheep mammary gland

The size distribution of methionyl-tRNA synthetase in extracts from sheep liver is compared to that of lysyl-tRNA, isoleucyl-tRNA, leucyl-tRNA and seryl-tRNA synthetases by gel filtration on Biogel A-5m. Extraction conditions are described which lead to isolation of methionyl-tRNA synthetase exclusively in the form of complexes of molecular weight close to 10(6). Limited trypsin treatment of these aggregates releases a fully active low-molecular-weight form of methionyl-tRNA synthetase which was purified to a specific activity of 674 units/mg at 25 degrees C with a yield of 40%. The homogeneous enzyme appears to be undistinguishable from the corresponding enzyme derived from sheep lactating mammary gland, as judged by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by titration with antibodies raised against the enzyme purified from liver.     

Purification and kinetic mechanism of lysyl-tRNA synthetase from Mycobacterium smegmatis SN2

Lysyl-tRNA synthetase [L-lys:tRNAlys ligase (AMP forming) EC:6.1.1.6] has been purified to homogeneity from Mycobacterium smegmatis SN2. The enzyme is a dimer of molecular weight 126,000 and is composed of identical subunits. A detailed analysis of the kinetic mechanism of the lysyl-tRNA synthetase has been carried out. A rapid equilibrium random ter ter mechanism is proposed based on initial velocity and product inhibition studies. There is no evidence for the formation of enzyme-bound lysyl-adenylate. The reverse reaction, studied by the deacylation of lysyl-tRNA, requires the presence of both AMP and PPi. This observation is consistent with the mechanism proposed.