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

Ej, Walker; Gb, Treacy; Pd, Jeffrey

doi:10.1021/bi00277a030

Cited by 28 publications

(13 citation statements)

References 20 publications

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“…None of the AI-CAH sera significantly (>30%) inhibited any synthetase activity, including seryl-tRNA synthetase (Table 1 and data not shown). Conversely, five autoimmune anti-aminoacyl-tRNA synthetase sera [anti-Jol (anti-histidyl-tRNA synthetase), anti-PL-7 (anti-threonyltRNA synthetase), anti-PL-12 (anti-alanyl-tRNA synthetase), anti-glycyl-tRNA synthetase, and anti-isoleucyl-tRNA synthetase] (6-10) showed reproducible >80% inhibition of the expected specific aminoacyl-tRNA synthetase activity relative to two normal human control sera (Table 1 and (14 (35), bovine liver extracts (36), and mouse cells (37) are all significantly larger (60-65 kDa) than this human antigen, and such divergence in size would not be expected. (iv) Most conclusively, the much more abundant major isoacceptor tRNAser species (38)(39)(40) are not coprecipitated by these AI-CAH sera, as would be expected for a synthetase antigen.…”

mentioning

confidence: 99%

Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion.

Gelpí

Sontheimer

Rodríguez-Sánchez

1992

Proc. Natl. Acad. Sci. U.S.A.

142

114

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We describe an autoantibody specificity present in a subgroup of patients with a severe form of autoimmune chronic active hepatitis. These antibodies precipitate a 90-nucleotide RNA from human whole cell extracts and recognize a 48-kDa polypeptide in immunoblotting assays. The RNA is a UGA suppressor serine tRNA that carries selenocysteine (tRNAlserJs'¢), as shown by sequence analysis. The protein does not appear to be seryl-tRNA synthetase; rather, it is an excellent candidate for a factor involved in cotranslational selenocysteine incorporation in human cells.Autoantibodies reactive with cellular constituents are often detected in sera from patients with autoimmune disease. Autoantibodies that immunoprecipitate tRNA have been detected primarily in sera from patients with polymyositis (1-3) and, less frequently, in patients with systemic lupus erythematosus (4) and scleroderma (5). The reactive antigens are usually tRNA-associated proteins; in only a few cases are they the tRNAs themselves (4, 6). Some of these protein antigens have been identified as aminoacyl-tRNA synthetases (3, 6-10).Other well-characterized components of the protein synthetic machinery [for example, ribosomes (11), 5S ribonucleoprotein (12), and elongation factor la (13)] also act as autoantigens. One translational event, the incorporation of selenocysteine into a growing polypeptide chain, is not well-understood in eukaryotes, and to our knowledge, the factors involved in this process have not been implicated in autoimmunity. The synthesis of selenoproteins in Escherichia coli involves several reactions (for review, see ref. 14). Briefly, selenocysteine incorporation is cotranslational and requires an unusual suppressor aminoacyl-tRNA (selenocysteyl-tRNAsec, where Sec is selenocysteine) and a specific translation factor (the product of the selB gene) that substitutes for elongation factor Tu. Insertion is directed by certain UGA triplets that in other contexts act as termination codons. tRNASec is initially charged with serine to form seryltRNASec and is then converted to selenocysteyl-tRNAsec by the action of the selA (selenocysteine synthase) and selD gene products. In mammalian cells, selenocysteine incorporation also occurs at specific UGA codons, and a suppressor serine tRNA that carries selenocysteine (tRNA[Serlsec) has been detected (15). However, complete elucidation of the eukaryotic pathway (and identification of the factors involved) has not yet been achieved.Here we report that an RNA-protein complex is the target of an autoantibody specificity found in the sera of a subset of patients with autoimmune chronic active hepatitis (AI-CAH). The RNA corresponds to a previously identified human suppressor serine tRNA, and the antigenic protein may be a factor involved in the pathway of cotranslational incorporation of selenocysteine into specific polypeptide chains.MATERIALS AND METHODS Subjects. Sera from 35 patients with AI-CAH were collected over a period of 4 years at the Sant Pau and Valle Hebron Hospitals in Barcelona. ...

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confidence: 99%

Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion.

Gelpí

Sontheimer

Rodríguez-Sánchez

1992

Proc. Natl. Acad. Sci. U.S.A.

142

114

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“…In extracts of higher eukaryotic cells, these enzymes are generally found in high molecular weight multienzyme complexes, often containing other components as well (2,3). The number of synthetases found in these complexes can vary depending on the method of isolation, but as many as eight or nine are routinely found associated with each other (4-7), and even higher numbers have been observed (8,9). It is now known that hydrophobic interactions play an important role in holding aminoacyltRNA synthetases in the complex (10)(11)(12) and that terminal extensions on the individual proteins, not required for catalysis, participate in these associations (13).…”

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confidence: 99%

Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for protein synthesis.

Sivaram

Deutscher

1990

Proc. Natl. Acad. Sci. U.S.A.

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Arginyl-tRNA synthetase (arginine-tRNA ligase, EC 6.1.1.19) is found in extracts of mammalian cells both as a free protein (Mr = 60,000) and as a component (Mr 72,000) of the high molecular weight aminoacyl-tRNA synthetase complex (Mr > 106). Several pieces of evidence indicate that the low molecular weight free form is not a proteolytic degradation product of the complex-bound enzyme but that it preexists in vivo: (i) the endogenous free form differs in size from the active proteolytic fragment generated in vitro, (ii) conditions expected to increase or decrease the amount of proteolysis do not alter the ratio of the two forms of the enzyme, and (iu) the free form contains an NH2-terminal methionine residue. A model is presented that provides a rationale for the existence of two forms of arginyl-tRNA synthetase in cells. In this model the complexed enzyme supplies arginyl-tRNA for protein synthesis, whereas the free enzyme provides arginyltRNA for the N112-terminal arginine modification of proteins by arginyl-tRNA:protein arginyltransferase. This latter process targets certain proteins for removal by the ubiquitindependent protein degradation pathway. The necessity for an additional pool of arginyl-tRNA for the modifcation reaction leads to the conclusion that the arginyl-tRNA destined for protein synthesis (and/or protein modification) is channeled and unavailable for other processes. Other evidence supporting channeling in protein synthesis is discussed.It is becoming increasingly clear that many, if not all, of the macromolecular components of cells are organized into multienzyme complexes or associated with subcellular structures (see ref. 1 for a review). A major consequence of such organization is that it can lead to compartmentalization or channeling of metabolic pathways (i.e., the transfer of metabolites from one enzyme to another without equilibration with the total fluid of the cell). Channeled pathways presumably would be more efficient than free ones by increasing the local substrate concentrations for a given amount of substrate, by

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“…It has been purified from several sources [2][3][4], and recently we have developed a rapid method for obtaining the human enzyme in high yield (5000-fold purified) from HeLa cells for immunological studies [5]. We have found, however, as had been reported for the enzyme from other sources [3,4], that it is markedly labile to dilution or storage. We sought, therefore, a method to preserve enzymatic activity for further studies.…”

Section: Introductionmentioning

confidence: 94%

Stimulation and partial stabilization of human histidyl‐tRNA synthetase by hemoglobin

1988

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Histidyl‐tRNA synthetase, an enzyme against which antibodies are directed in some patients with polymyositis, has been purified 5000‐fold from HeLa cells, but was extremely labile to dilution or on storage at −80°C. In order to facilitate study of the biochemical and immunological properties of the enzyme, a stabilizer was sought. Hemoglobin at 2 mg/ml was found to stimulate the enzyme and also partially preserved the activity of the enzyme stored at a low concentration (< 10μg/ml). Hematin, but not the globin protein, could substitute for hemoglobin in stimulating the enzyme.

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

Cited by 28 publications

References 20 publications

Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion.

Autoantibodies against a serine tRNA-protein complex implicated in cotranslational selenocysteine insertion.

Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for protein synthesis.

Stimulation and partial stabilization of human histidyl‐tRNA synthetase by hemoglobin

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