The role of cyclic 3',5'-AMP in the regulation of aminoacyl-tRNA synthetase activities in mouse uterus and liver following 17beta-oestradiol treatment. Activation of a phosphoaminoacyl-tRNA synthetase phosphatase by phosphorylation with cyclic 3',5'-AMP dependent protein kinase.

The changes in the activities of 17 aminoacyl-tRNA synthetases induced by phosphorylation [1] were reversed by the action of cyclic AMP in preparations from both uterus and liver. Cyclic AMP also inhibited the phosphorylation of aminoacyl-tRNA synthetase protein by endogenous non-cyclic AMP-dependent protein kinase and [gamma-32P]ATP. The effect was not due to a stimulation of phosphoaminoacyl-tRNA synthetase phosphatase or to an influence of cyclic AMP on aminoacyl-tRNA synthetases. The activity of phosphoaminoacyl-tRNA synthetase phosphatase was increased by treatment with endogenous cyclic AMP-dependent protein kinase, ATP and cyclic AMP. Affinity chromatography of the 32P-labeled phosphorylated phosphosynthetase phosphatase protein followed by gel electrophoresis showed that the activated phosphatase was phosphorylated. In the uterus, the changes in 17 aminoacyl-tRNA synthetase activities observed 5 min after dibutyryl cyclic AMP administration to ovariectomized mice were similar to those observed after 17beta-oestradiol treatment, whereas in the liver the changes in these activities were the opposite to those found after treatment with 17beta-oestradiol. A mechanism for the regulation of the 17 aminoacyl-tRNA synthetase activities is proposed, which suggests that the synthetase activities inhibited (group I) or stimulated (group II) by phosphorylation with a non-cyclic AMP-dependent aminoacyl-tRNA synthetase kinase are reactivated (group I) or inhibited (group II), respectively, by the action of a cyclic AMP-dependent phosphatase kinase through the increased activity of phosphorylated phosphoaminoacyl-tRNA synthetase phosphatase.     

Evidence that lysyl- and/or arginyl-tRNA synthetases from rat liver contain carbohydrate.

Lysyl- and arginyl-tRNA synthetases have been found to exist in multiple molecular weight forms in rat liver. The small molecular weight forms of lysyl- and arginyl-tRNA synthetases copurify throughout a five step chromatographic procedure resulting in a purification of 370- and 140-fold, respectively. The enzymes appear to be homogeneous on Sephadex G-200 and elute at an apparent molecular weight of 240,000. Gas chromatography reveals that the synthetases contain nearly 14% carbohydrate by weight. The carbohydrates present are: mannose, fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. This is the first report that aminoacyl-tRNA synthetases may exist as glyco-proteins.     

The activity and sedimentation properties of the aminoacyl-tRNA synthetases of rat skeletal muscle.

The aminoacyl-tRNA synthetases of the postribosomal supernatant fraction of rat skeletal muscle were characterized by their activity and sedimentation properties. The synthetases of muscle were compared with those of liver in terms of these parameters. Extraction of the synthetases of muscle with a buffer containing 4 mM adenosine triphosphate (ATP) resulted in a 50--100% increase in the activities of glutaminyl-, glutamyl-, isoleucyl-, leucyl-, lysyl-, methionyl-, and prolyl-tRNA synthetases in the postribosomal fraction, over those activities extracted in the absence of ATP. This effect of ATP was specific for those synthetases which sedimented as particulate elements in sucrose gradients, and appeared to be unique to muscle. The individual synthetase activities of muscle, except alanyl-, leucyl-, and valyl-tRNA synthetases, were aprrox. 25% of the corresponding synthetase activities of liver. Sucrose density gradient analysis of the postribosomal fraction of muscle and liver revealed that the sedimentation profiles of the synthetases of the two tissues were similar, with nine synthetase activities sedimenting as large particulate entities at 18 S. The findings suggest that the particulate forms of the synthetases reflect true association of the enzymes with a high molecular weight cellular component common to both tissues.     

I. A study of the stages in the quantitative isolation of aminoacyl-tRNA synthetase activities from mouse liver.

The elution profiles of 17 aminoacyl-tRNA synthetases from chromatography of 149 000 x g supernatant on Sephadex G-200 were determined as well as the influence of different methods of homogenization and of chromatography on DEAE-cellulose on the elution profiles. With gentle homogenization all synthetases were eluted in the void volume in four different peaks, containing (a) leucyl- and phenylalanyl-, (b) lysyl-, prolyl-, isoleucyl-, methionyl-, glycl-, and valyl-, (c) arginyl-, alanyl-, and asparaginyl- and (d) aspartyl-, histidyl-, seryl-, threonyl-, glutaminyl-, and tyrosyl- tRNA synthetases. With less gentle homogenization, peaks of lower molecular weight appeared. More than two peaks for each aminoacyl-tRNA synthetases were never found. Of the aminoacyl-tRNA synthetases examined, alanyl-,arginyl-, aspartyl-, leucyl- and lysyl-tRNA synthetases were not inactivated by chromatography on DEAE-cellulose, whereas phenylalanyl- and seryl-tRNA synthetases lost 60% of their activity.     

Disassembly and gross structure of particulate aminoacyl-tRNA synthetases from rat liver. Isolation and the structural relationship of synthetase complexes.

The major high molecular weight complex of aminoacyl-tRNA synthetases is purified about 1000-fold with 30% yield from rat liver. The synthetase complex sediments at 24 S with a molecular weight of 900,000 +/- 75,000 and contains aminoacylation activities for lysine, arginine, isoleucine, leucine, methionine, glutamine, glutamate, and proline. The 24 S synthetase complex dissociates into 21 S, 18 S, 13 S, 12 S, and 10 S complexes with specific enzymatic activities. Dissociation of the 24 S complex into active free synthetases is achieved by hydrophobic interaction chromatography. The disassembly of the synthetase complex is consistent with the structural model of a heterotypic multienzyme complex and suggests that the complex formation is due to the specific intermolecular interactions among the synthetases.     

Occurrence of aminoacyl-tRNA synthetase complexes in calf brain

Eighteen aminoacyl-tRNA synthetases of the postribosomal supernatant fraction of brain cortex were characterized by glycerol density gradient centrifugation and gel filtration analysis. On the basis of sedimentation properties and gel elution profiles, four groups of enzyme activities were determined in the postribosomal supernatant fraction; the first group sedimenting at about 6 S contained 18 individual synthetase activities, the next successive groups of greater molecular sizes contained synthetase complexes, and the last group possessed activities of 15 synthetases. Each aminoacyl-tRNA synthetase appeared at least in two forms: free and bound in complexes of varying sizes and different enzyme compositions. Conventional purification methods of lysyl-tRNA synthetase from the post-ribosomal supernatant fraction of brain cortex gave a preparation containing four groups of aminoacylation activities. The obtained preparation contained a large complex, reduced number of intermediate complexes and some individual synthetases.     

Aminoacyl-tRNA synthetase stimulatory factors and inorganic pyrophosphatase.

A protein was purified from rat liver which stimulated a number of liver aminoacyl-tRNA synthetases. This stimulatory factor was identical with the "tRNA activator" of Dickman & Boll [(1976) Biochemistry 15, 3925] in its mechanism of action and chemical properties, although it was considerably more purified. The two preparations stimulated synthetases by virtue of their pyrophosphatase activity which destroyed the potent inhibitor, PPi, that was present in the reaction mixtures. This PPi was either generated during the reaction or was introduced by contamination of the tRNA or ATP preparations. The degree of inhibition of PPi was strongly influenced by assay conditions, being most effective at low amino acid concentrations, at low pH, and in the presence of heterologous tRNAs. By use of certain assay conditions, PPi concentrations as low as 2 microM could inhibit some synthetases close to 50%. The pitfalls associated with some assay conditions commonly used for aminoacyl-tRNA synthetases are discussed. These studies raise questions about the physiological significance of many previously described aminoacyl-tRNA synthetase stimulatory factors.     

The influence of 17-beta-estradiol 17-beta-acetate on adenylyl cyclase activity and aminoacyl-tRNA synthetase phosphatase activity in ovariectomized NMRI mice.

Adenylyl cyclase activity in the uterus of ovariectomized NMRI mice showed an increase of 87% four minutes after intraperitoneal administration of 17-beta-estradiol 17-beta-acetate in water solution, while in the liver of the same animals activity had decreased by 57%. At the same time, the activity of the 51 kDa form of the aminoacyl-tRNA synthetase phosphatase in the uterus showed an increase of 400%, while in the liver a decrease of 60% was observed. Sixty minutes after hormone injection the adenylyl cyclase activities showed no difference from controls.     

Chromatofocusing of aminoacyl-tRNA synthetases, extracted from NMRI mouse liver

Chromatofocusing of 17 aminoacyl-tRNA synthetases extracted from NMRI mouse liver is described and the apparent isoelectric points of these enzymes are presented. Each of 15 aminoacyl-tRNA synthetases was present in two peaks. Isoleucyl-tRNA synthetase showed only one peak and arginyl-tRNA synthetase was present in three peaks. Phosphorylation/dephosphorylation experiments with arginyl-tRNA synthetase indicate that the peaks represent phosphorylated and unphosphorylated synthetase protein. One example of detection of increased protein phosphorylation during a biological experiment is presented.     

Aminoacyl-tRNA-synthetases and their high molecular weight complexes in experimental myocardial ischemia

A number of aminoacyl-tRNA synthetases from rabbit liver during experimental myocardial infarction and from pig myocardium upon 15-min of autolysis were found to increase their activity in aminoacylation. Direct correlations between the activities of high molecular weight complexes and of the total extracts were not observed. It was shown that the specific activity of endogenous inorganic pyrophosphatase increased markedly during the ischemia of myocardium both in total myocardium extracts and in high molecular weight complexes.     

Association of eukaryotic aminoacyl-tRNA-synthases with polyribosomes

Upon fractionation of a mitochondria-free extract of rabbit reticulocytes into a ribosome-free extract and mono- and polyribosomes the bulk of the aminoacyl-tRNA synthetase activity was found in the fraction of mono- and polyribosomes. All the fifteen aminoacyl-tRNA synthetases were revealed, although in somewhat different quantities, in both fractions of the mitochondria-free reticulocyte extract. Aminoacyl-tRNA synthetases of the ribosome-free extract are found in two forms: RNA-binding one, and, the one having no affinity for high molecular weight RNAs. Aminoacyl-tRNA synthetases dissociated from the complexes with polyribosomes exist only in the RNA-binding form. All aminoacyl-tRNA synthetases can be removed from such complexes by an addition of 16S rRNA of E. coli, poly(U) or tRNA of rabbit reticulocytes. This testifies to labile association of aminoacyl-tRNA synthetases with the RNA-component of polyribosomes as well as to a rather nonspecific character of their interaction. After EDTA-induced dissociation of polyribosomes, the aminoacyl-tRNA synthetase activity was detected in the complex with both ribosomal subunits.     

Initial position of aminoacylation of individual Escherichia coli, yeast, and calf liver transfer RNAs.

Transfer RNAs from Escherichia coli, yeast (Sacharomyces cerevisiae), and calf liver were subjected to controlled hydrolysis with venom exonuclease to remove 3'-terminal nucleotides, and then reconstructed successively with cytosine triphosphate (CTP) and 2'- or 3'-deoxyadenosine 5'-triphosphate in the presence of yeast CTP(ATP):tRNA nucleotidyltransferase. The modified tRNAs were purified by chromatography on DBAE-cellulose or acetylated DBAE-cellulose and then utilized in tRNA aminoacylation experiments in the presence of the homologous aminoacyl-tRNA synthetase activities. The E. coli, yeast, and calf liver aminoacyl-tRNA synthetases specific for alanine, glycine, histidine, lysine, serine, and threonine, as well as the E. coli and yeast prolyl-tRNA synthetases and the yeast glutaminyl-tRNA synthetase utilized only those homologous modified tRNAs terminating in 2'-deoxyadenosine (i.e., having an available 3'-OH group). This is interpreted as evidence that these aminoacyl-tRNA synthetases normally aminoacylate their unmodified cognate tRNAs on the 3'-OH group. The aminoacyl-tRNA synthetases from all three sources specific argining, isoleucine, leucine, phenylalanine, and valine, as well as the E. coli and yeast enzymes specific for methionine and the E. coli glutamyl-tRNA synthetase, used as substrates exclusively those tRNAs terminating in 3'-deoxyadenosine. Certain aminoacyl-tRNA synthetases, including the E. coli, yeast, and calf liver asparagine and tyrosine activating enzymes, the E. coli and yeast cysteinyl-tRNA synthetases, and the aspartyl-tRNA synthetase from yeast, utilized both isomeric tRNAs as substrates, although generally not at the same rate. While the calf liver aspartyl- and cysteinyl-tRNA synthetases utilized only the corresponding modified tRNA species terminating in 2'-deoxyadenosine, the use of a more concentrated enzyme preparation might well result in aminoacylation of the isomeric species. The one tRNA for which positional specificity does seem to have changed during evolution is tryptophan, whose E. coli aminoacyl-tRNA synthetase utilized predominantly the cognate tRNA terminating in 3'-deoxyadenosine, while the corresponding yeast and calf liver enzymes were found to utilize predominantly the isomeric tRNAs terminating in 2'-deoxyadenosine. The data presented indicate that while there is considerable diversity in the initial position of aminoacylation of individual tRNA isoacceptors derived from a single source, positional specificity has generally been conserved during the evolution from a prokaryotic to mammalian organism.     

Multiple forms of arginyl- and lysyl-tRNA synthetases in rat liver: a re-evaluation

The size distribution of lysyl- and arginyl-tRNA synthetases in crude extracts from rat liver was re-examined by gel filtration. It is shown that irrespective of the addition or not of several proteinase inhibitors, lysyl-tRNA synthetase was present exclusively as a high-Mr entity, while arginyl-tRNA synthetase occurred as high- and low-Mr forms, in the constant proportions of 2:1, respectively. The polypeptide molecular weights of the arginyl-tRNA synthetase in these two forms were 74000 and 60000, respectively. The high-Mr forms of lysyl- and arginyl-tRNA synthetases were co-purified to yield a multienzyme complex, the polypeptide composition of which was virtually identical to that of the complexes from rabbit liver and from cultured Chinese hamster ovary cells. Of the nine aminoacyl-tRNA synthetases, specific for lysine, arginine, methionine, leucine, isoleucine, glutamine, glutamic and aspartic acids and proline, which characterize the purified complex, each, except prolyl-tRNA synthetase, was assigned to the constituent polypeptides by the protein-blotting procedure, using the previously characterized antibodies to the aminoacyl-tRNA synthetase components of the corresponding complex from sheep liver.     

Macromolecular complexes of aminoacyl-tRNA synthetases from eukaryotes. 1. Extensive purification and characterization of the high-molecular-weight complex(es) of seven aminoacyl-tRNA synthetases from sheep liver.

Starting from homogenates of sheep liver, extensive co-purification of seven aminoacyl-tRNA synthetases to high specific activities was achieved by a three-step procedure involving fractional precipitation by poly(ethylene glycol) 6000, gel filtration on 6% agarose and chromatography on Sepharose-bound tRNA. The purified material is composed of nine major protein components as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and has an apparent molecular weight of about 10(6) estimated by gel filtration on 6% agarose. It contains aminoacyl-tRNA synthetase activities specific for methionine, lysine, arginine, leucine, isoleucine, glutamine and glutamic acid. The rigorous co-elution of these seven enzymes at each chromatographic step suggests, but does not conclusively prove, that they are physically associated within the same complex. The enzyme composition of the high-molecular-weight complex purified from sheep liver is identical to that of the complex previously isolated from human placenta by Denney in 1977 (Arch. Biochem. Biophys. 183, 156--167).     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sedimentation behaviour of aminoacyl-tRNA synthetases from mixed lysates of yeast and rabbit liver

The subcellular distribution of five aminoacyl-tRNA synthetases from yeast, including lysyl-, arginyl- and methionyl-tRNA synthetases known to exist as high-molecular-weight complexes in lysates from higher eukaryotes, was investigated. To minimize the risks of proteolysis, spheroplasts prepared from exponentially grown yeast cells were lysed in the presence of several proteinase inhibitors, under conditions which preserved the integrity of the proteinase-rich vacuoles. The vacuole-free supernatant was subjected to sucrose density gradient centrifugation. No evidence for multimolecular associations of these enzymes was found. In particular, phenylalanyl-tRNA synthetase activity was not associated with the ribosomes, whereas purified phenylalanyl-tRNA synthetase from sheep liver, added to the yeast lysate prior to centrifugation, was entirely recovered in the ribosomal fraction. A mixture of lysates from yeast and rabbit liver was also subjected to sucrose gradient centrifugation and assayed for methionyl- and arginyl-tRNA synthetase activities, under conditions which allowed discrimination between the enzymes originating from yeast and rabbit. The two enzymes from rabbit liver were found to sediment exclusively as high-molecular-weight complexes, in contrast to the corresponding enzymes from yeast, which displayed sedimentation properties characteristic of free enzymes. The preservation of the complexed forms of mammalian aminoacyl-tRNA synthetases upon mixing of yeast and rabbit liver extracts argues against the possibility that failure to observe complexed forms of these enzymes in yeast was due to uncontrolled proteolysis. Furthermore, this result denies the presence, in the crude extract from liver, of components capable of inducing artefactual aggregation of the yeast aminoacyl-tRNA synthetases, and thus indirectly argues against an artefactual origin of the multienzyme complexes encountered in lysates from mammalian cells.     

Molecular weights of mitochondrial and cytoplasmic aminoacyl-tRNA synthetases of beef liver and their complexes

In eukaryotes, multienzyme complexes containing five to nine aminoacyl-tRNA synthetase activities have frequently been reported. In this study, we report the existence, in bovine liver cytoplasm, of a multienzyme complex containing at least 16 activities which can be disrupted by homogenization to give rise to smaller complexes and noncomplexed synthetases. Determination of the size and component activity of these complexes and of the molecular weights of all 20 free synthetases suggests that the smaller complexes and free activities normally identified arise from the larger complex by well-defined stages during homogenization. We also show that similar, though not identical, complexes are found in bovine liver mitochondria and give the molecular weights of 16 mitochondrial synthetases.     

High-molecular-weight forms of aminoacyl-tRNA synthetases and tRNA modification enzymes in Escherichia coli.

The presence of high-molecular-weight complexes of aminoacyl-tRNA synthetases in Escherichia coli has been reported (C. L. Harris, J. Bacteriol. 169:2718-2723, 1987). In the current study, Bio-Gel A-5M gel chromatography of 105,000 x g supernatant preparations from E. coli Q13 indicated high molecular weights for both tRNA methylase (300,000) and tRNA sulfurtransferase (450,000). These tRNA modification enzymes did not appear to exist in the same multienzymic complex. On the other hand, 4-thiouridine sulfurtransferase eluted with aminoacyl-tRNA synthetase activity on Bio-Gel A-5M, and both of these activities were cosedimented after further centrifugation of cell supernatants at 160,000 x g for 18 h. Despite this evidence for association of the sulfurtransferase with the synthetase complex, isoleucyl-tRNA synthetase and tRNA sulfurtransferase were totally resolved from each other by DEAE-Sephacel chromatography. Subsequent gel chromatography showed little change in their elution positions on agarose. Hence, either nonspecific aggregation occurred here, or the modification enzymes studied are not members of the aminoacyl-tRNA synthetase complex in E. coli. These findings do suggest that some bacterial tRNA modification enzymes are present in multiprotein complexes of high molecular weight.     

Studies on transfer RNA from mycobacteria.

Active preparations of tRNA and aminoacyl-tRNA synthetases have been isolated from exponentially growing cells of Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. Though the aminoacyl-tRNA synthetases of older cells retain their activity, the tRNAs seem to undergo modification and show poorer activity. The mycobacterial enzyme preparations catalyse homologous and heterologous aminoacylation between tRNA from the two species (M. smegmatis and M. tuberculosis H37Rv) or from Escherichia coli, with equal efficiency; tRNA samples from eukaryotic cells (yeast and rat liver) do not serve as substrates for the mycobacterial synthetases. The analytical separation of the different amino acid specific tRNAs from M. smegmatis resembles the pattern found in other bacteria. Purification of valine- (three species) and methionine-specific tRNA (two species) to 70-80% purity has been accomplished by using column-chromatographic techniques. Of the two species of tRNAMet, one can be formylated in the presence of formyl tetrahydrofolate and the transformylase from mycobacteria.     

Isolation and electron microscopic characterization of the high molecular mass aminoacyl-tRNA synthetase complex from murine erythroleukemia cells

A high molecular mass aminoacyl-tRNA synthetase complex has been isolated from a murine erythroleukemia cell line. This multienzyme complex contains activities for the arginyl-, aspartyl-, glutamyl-, glutaminyl-, isoleucyl,- leucyl-, lysyl-, methionyl-, and prolyl-tRNA synthetases. This enzyme composition, the polypeptide pattern observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the relative stoichiometry of the component polypeptides are characteristic of high molecular mass complexes of aminoacyl-tRNA synthetases isolated from a variety of mammalian tissues and cell types. Negatively stained preparations of native complex and of glutaraldehyde-treated material have been examined by electron microscopy. In both cases, a distinctive particle is observed which appears in several orientations. The most common views are of two different projections of a squarish particle that measures approximately 27 x 27 nm. Other commonly observed views are of a "U" shape, a rectangle, and a triangle. All of these views are seen in both gradient-purified samples and those prepared directly from material as isolated. These data are consistent with a model for the multienzyme aminoacyl-tRNA synthetase complex as a "cup" or elongated U structure. These studies demonstrate that the high molecular mass complex of eukaryotic aminoacyl-tRNA synthetases does have a coherent structure that can be visualized by electron microscopy.