Protective mechanisms against homocysteine toxicity: the role of bleomycin hydrolase

Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.     

Identification and origin of Nε-homocysteinyl-lysine isopeptide in humans and mice

Homocysteine (Hcy) metabolites, Hcy-thiolactone and N-Hcy-proteins, have been linked to the pathology of human cardiovascular and neurodegenerative diseases. Hcy-thiolactone is generated in an error-editing reaction in protein biosynthesis when Hcy is selected in place of methionine by methionyl-tRNA synthetase. N-Hcy-protein, in which Hcy is linked via isopeptide bond to ε-amino group of a protein lysine residue, forms in a post-translational reaction of Hcy-thiolactone with proteins. Here, we identify a novel metabolite, Nε-Hcy-Lys, in human and mouse plasma, and show that this metabolite is elevated in genetic (cystathionine β-synthase deficiency in humans and mice, methylenetetrahydrofolate reductase deficiency in mice) or dietary (high Met diet in mice) deficiencies in Hcy metabolism. We also show that Nε-Hcy-Lys is generated by proteolytic degradation of N-Hcy-protein in mouse liver extracts. Our data indicate that free Nε-Hcy-Lys is an important pathology-related component of Hcy metabolism in humans and mice.     

Quantification of urinary S- and N-homocysteinylated protein and homocysteine-thiolactone in mice

Homocysteine (Hcy) and its metabolites Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein are implicated in vascular and neurological diseases. However, quantification of these metabolites remains challenging. Here I describe streamlined assays for these metabolites based on their conversion to Hcy-thiolactone. Free Hcy-thiolactone is extracted from the urine with chloroform/methanol. Total Hcy is converted to Hcy-thiolactone in the presence of 1 N HCl. Major urinary protein (MUP)-bound S-linked Hcy is liberated from the protein by reduction with dithiothreitol and converted to Hcy-thiolactone. Acid hydrolysis of MUP with 6 N HCl liberates N-linked Hcy as Hcy-thiolactone, which is then extracted with chloroform/methanol. Ferritin is used as an N-Hcy-protein standard and an authentic Hcy-thiolactone is used to monitor the efficiency of extraction. Hcy-thiolactone (free, derived from total Hcy, or from MUP-bound N-linked or S-linked Hcy) is separated by a cation exchange high-performance liquid chromatography, post-column derivatized with o-phthaldialdehyde, and quantified by fluorescence. Using these assays with as little as 2–20 μL of urine I show that MUP carry N-linked and S-linked Hcy and that N-Hcy-MUP and S-Hcy-MUP and Hcy-thiolactone are severely elevated in cystathionine β-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease.     

Translational incorporation of S-nitrosohomocysteine into protein

The non-protein amino acid homocysteine (Hcy), owing to its structural similarity to the protein amino acids methionine, isoleucine, and leucine, enters first steps of protein synthesis and is activated by methionyl-, isoleucyl-, and leucyl-tRNA synthetases in vivo. However, translational incorporation of Hcy into protein is prevented by editing mechanisms of these synthetases, which convert misactivated Hcy into thiolactone. The lack of efficient interactions of the side chain of Hcy with the specificity subsite of the synthetic/editing active site is a prerequisite for editing of Hcy. Thus, if the side chain thiol of Hcy were reversibly modified with a small molecule that would enhance its binding to the specificity subsite and prevent editing, such modified Hcy is predicted to be transferred to tRNA and incorporated translationally into protein. Here I show that S-nitroso-Hcy is in fact transferred to tRNA by methionyl-tRNA synthetase and incorporated into protein by the bacterium Escherichia coli. S-Nitroso-Hcy-tRNA also supports translation of mRNAs in a rabbit reticulocyte system. Removal of the nitroso group yields Hcy-tRNA and protein containing Hcy in peptide bonds. S-Nitrosylation-mediated translational incorporation of Hcy into protein may occur under natural conditions in cells and contribute to Hcy-induced pathogenesis in atherosclerosis.     

tRNAfMet-induced conformational transition at the intersubunit domain of fluorescent-labeled methionyl-tRNA synthetase

Conformational transition in methionyl-tRNA synthetase upon binding of tRNAfMet, whose binding shows strong negative cooporativity, was analyzed by fluorescence spectroscopy. The fluorescent probe N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS) reacts with native methionyl-tRNA synthetase in a nearly stoichiometric amount (2 per dimer) without affecting enzyme activity. The probe is shown by controlled trypsinization to be located in a 130 amino acid fragment at the C-terminus joining the subunits. The emission and excitation spectra, rotational freedom, and solvent accessibility of the fluorophore in AEDANS-methionyl-tRNA synthetase are analyzed. The results suggest that the probe is localized in a nonpolar environment, nearly immobile relative to methionyl-tRNA synthetase yet fully accessible to the solvent. Upon binding of tRNAfMet, the fluorescence intensity in AEDANS-methionyl-tRNA synthetase was appreciably reduced without a shift in the emission or excitation spectra. Lifetime measurement shows that a static mechanism accounts for the observed quenching. Furthermore, the remaining emitting AEDANS becomes effectively shielded from solvent molecules. These results suggest an unsymmetric conformational transition at the intersubunit domains of the two subunits in methionyl-tRNA synthetase upon binding one molecule of tRNAfMet.     

Initiation of in vivo protein synthesis with non-methionine amino acids

Methionine is the universal amino acid for initiation of protein synthesis in all known organisms. The amino acid is coupled to a specific initiator methionine tRNA by methionyl-tRNA synthetase. In Escherichia coli, attachment of methionine to the initiator tRNA (tRNA(fMet)) has been shown to be dependent on synthetase recognition of the methionine anticodon CAU (complementary to the initiation codon AUG), [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759]. We show here that alteration of the anticodon of tRNA(fMet) to GAC or GAA leads to aminoacylation of the initiator tRNA with valine or phenylalanine. In addition, tRNA(fMet) carrying these amino acids initiates in vivo protein synthesis when provided with initiation codons complementary to the modified anticodons. These results indicate that the sequence of the anticodon of tRNA(fMet) dictates the identity of the amino acid attached to the initiator tRNA in vivo and that there are no subsequent steps which prevent initiation of E. coli protein synthesis by valine and phenylalanine. The methods described here also provide a convenient in vivo assay for further examination of the role of the anticodon in tRNA amino acid acceptor identity.     

Homocysteine thiolactone and <Emphasis Type="Italic">N</Emphasis>-homocysteinylated protein induce pro-atherogenic changes in gene expression in human vascular endothelial cells

Genetic or nutritional deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis. In addition to Hcy, related metabolites accumulate in HHcy but their role in endothelial dysfunction is unknown. Here, we examine how Hcy-thiolactone, N-Hcy-protein, and Hcy affect gene expression and molecular pathways in human umbilical vein endothelial cells. We used microarray technology, real-time quantitative polymerase chain reaction, and bioinformatic analysis with PANTHER, DAVID, and Ingenuity Pathway Analysis (IPA) resources. We identified 47, 113, and 30 mRNAs regulated by N-Hcy-protein, Hcy-thiolactone, and Hcy, respectively, and found that each metabolite induced a unique pattern of gene expression. Top molecular pathways affected by Hcy-thiolactone were chromatin organization, one-carbon metabolism, and lipid-related processes [?log(P value) = 20–31]. Top pathways affected by N-Hcy-protein and Hcy were blood coagulation, sulfur amino acid metabolism, and lipid metabolism [?log(P value)] = 4–11; also affected by Hcy-thiolactone, [?log(P value) = 8–14]. Top disease related to Hcy-thiolactone, N-Hcy-protein, and Hcy was ‘atherosclerosis, coronary heart disease’ [?log(P value) = 9–16]. Top-scored biological networks affected by Hcy-thiolactone (score = 34–40) were cardiovascular disease and function; those affected by N-Hcy-protein (score = 24–35) were ‘small molecule biochemistry, neurological disease,’ and ‘cardiovascular system development and function’; and those affected by Hcy (score = 25–37) were ‘amino acid metabolism, lipid metabolism,’ ‘cellular movement, and cardiovascular and nervous system development and function.’ These results indicate that each Hcy metabolite uniquely modulates gene expression in pathways important for vascular homeostasis and identify new genes and pathways that are linked to HHcy-induced endothelial dysfunction and vascular disease.     

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone.

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.     

Metabolism and neurotoxicity of homocysteine thiolactone in mice: protective role of bleomycin hydrolase

Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze L-Hcy-thiolactone (but not D-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh (-/-) mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh (+/+) animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh (-/-) mice relative to Blmh (+/+) animals. Plasma N-Hcy-protein was elevated in Blmh (-/-) mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh (+/+) animals. More intraperitoneally injected L-Hcy-thiolactone was recovered in plasma in Blmh (-/-) mice than in wild-type Blmh (+/+) animals (83.1 vs. 39.3 μM, P < 0.0001). In Blmh (+/+) mice injected intraperitoneally with D-Hcy-thiolactone, D,L-Hcy-thiolactone, or L-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by L-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh (-/-) than in Blmh (+/+) mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against L-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.     

Deletion analysis in the amino-terminal extension of methionyl-tRNA synthetase from Saccharomyces cerevisiae shows that a small region is important for the activity and stability of the enzyme

MESI, the structural gene for methionyl-tRNA synthetase from Saccharomyces cerevisiae encodes an amino-terminal extension of 193 amino acids, based on the comparison of the encoded protein with the Escherichia coli methionyl-tRNA synthetase. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA aminoacylation step than on the adenylate formation step. The Km for ATP and methionine are unaltered indicating that the global structure of the enzyme is maintained. The Km for tRNA increased slightly by a factor of 3 which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the Vmax of the enzyme. Examination of the three-dimension structure of the homologous E. coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide-binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagating a mutational alteration to the catalytic residues.     

Regulation of Synthesis of Methionyl-, Prolyl-, and Threonyl-Transfer Ribonucleic Acid Synthetases of Escherichia coli

Proline- and threonine-restricted growth caused a three- to fourfold derepression of the differential rate of synthesis of the prolyl- and threonyl-transfer ribonucleic acid (tRNA) synthetases, respectively. Similarly, there was approximately a 24-fold derepression in the rate of synthesis of methionyl-tRNA synthetase during methionine restriction. Addition of the respective amino acids to such derepressed cultures resulted in a repression of synthesis of their cognate synthetases. These results support previous findings and further strengthen the idea that the formation of aminoacyl-tRNA synthetases is regulated by some mechanism which is mediated by the cognate amino acids.     

Modification by homocysteine thiolactone affects redox status of cytochrome C

Homocysteine (Hcy)-thiolactone mediates a post-translational incorporation of Hcy into protein in humans. Protein N-homocysteinylation is detrimental to protein structure and function and is linked to pathophysiology of hyperhomocysteinemia observed in humans and experimental animals. The modification by Hcy-thiolactone can be detrimental directly by affecting the function of an essential lysine residue or indirectly by interfering with the function of other essential residues or cofactors. Previous work has shown that cytochrome c is very sensitive to Hcy-thiolactone, which causes formation of N-Hcy-cytochrome c multimers. However, it was unclear what sites in cytochrome c were prone to Hcy attachment and whether N-linked Hcy can affect the structure and redox function of cytochrome c. Here we show that 4 lysine residues (Lys8 or -13, Lys86 or -87, Lys99, and Lys100) of cytochrome c are susceptible to N-homocysteinylation. We also show that N-homocysteinylation of 1 mol of lysine/mol of protein affects the redox state of the heme ligand of cytochrome c by rendering it reduced. The modification causes subtle structural changes, manifested as increased resistance of the N-Hcy-cytochrome c to proteolysis by trypsin, chymotrypsin, and Pronase. However, no major secondary structure perturbations were observed as shown by circular dichroism spectroscopy. Our data illustrate how N-homocysteinylation can interfere with the function of heme-containing proteins.     

Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases from an extreme thermophile Thermus thermophilus HB8: protein structure and Zn2+ binding

Thermostable valyl-tRNA, isoleucyl-tRNA and methionyl-tRNA synthetases have been purified from an extreme thermophile, Thermus thermophilus HB8. Valyl-tRNA and isoleucyl-tRNA synthetases are found to be monomer proteins (Mr 108000 and 129000, respectively), while methionyl-tRNA synthetase is a dimer protein (Mr 150000). These enzymes are very similar with respect to amino acid compositions and alpha-helix contents as estimated by circular dichroism analyses. Furthermore, two Zn2+ are tightly bound to each of these synthetases. These data suggest that valyl-tRNA and isoleucyl-tRNA synthetases consist of two domains, each corresponding to the subunit of methionyl-tRNA synthetase.     

Enhanced level and metabolic regulation of methionyl-transfer ribonucleic acid synthetase in different strains of Escherichia coli K-12.

The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the structural gene of this enzyme was investigated. No structural alteration of this enzyme was observed in three eductants examined. These were isolated from strain AB311, which had a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. In two of the three eductants studied, the level of this enzyme was twofold higher than in their parental strain regardless of growth conditions used. In contrast, isoleucyl-, leucyl-, and valyl-tRNA synthetases had similar levels in all strains examined. Like valyl-tRNA synthetase, but to a lesser extent, methionyl-tRNA synthetase was subject to metabolic regulation. Coupling between the level of methionyl-tRNA synthetase and growth rate was observed even in strains that had an enhanced level of methionyl-tRNA synthetase. These results suggest that the formation of methionyl-tRNA synthetase remains subject to metabolic regulation even when the repression-like mechanism that controls the synthesis of this enzyme is altered. In addition, we report that in the merodiploid strain EM20031, which was haploid for the valyl-tRNA synthetase structural gene and diploid for the structural genes of methionyl-tRNA synthetase and D-serine deaminase, the levels of these latter two enzymes varied to a minor yet significant extent with the phosphate concentration of the culture medium; under the same conditions, the level of valyl-tRNA synthetase remained unchanged. Moreover, no variation of the levels of these three enzymes in response to phosphate was observed in the haploid strain HfrH. These results indicate that in the merodiploid strain EM20031, which carries the episome F32, the number of episomes per chromosome varies to some extent according to the phosphate concentration of the culture medium.     

Initiation of protein synthesis by folate-sufficient and folate-deficient Streptococcus faecalis R: partial purification and properties of methionyl-transfer ribonucleic acid synthetase and methionyl-transfer ribonucleic acid formyltransferase

The initiation of protein synthesis by Streptococcus faecalis R grown in folate-free culture occurs without N-formylation or N-acylation of methionyl-tRNA(f) (Met). Methionyl-tRNA synthetase and methionyl-tRNA formyltransferase were partially purified from S. faecalis grown under normal culture conditions in the presence of folate (plus-folate); the general properties of the enzymes were determined and compared with the properties of the enzymes purified from wild-type cells grown in the absence of folate (minus-folate). S. faecalis methionyl-tRNA synthetase displays optimal activity at pH values between 7.2 and 7.8, requires Mg(2+), and has an apparent molecular weight of 106,000, as determined by gel filtration, and 127,000, as determined by sucrose density gradient centrifugation. The K(m) values of plus-folate methionyl-tRNA synthetase for each of the three substrates in the aminoacylation reaction (l-methionine, adenosine triphosphate, and tRNA) are nearly identical to the respective substrate Michaelis constants of minus-folate methionyl-tRNA synthetase. Furthermore, both plus- and minus-folate S. faecalis methionyl-tRNA synthetases catalyze, at equal rates, the aminoacylation of tRNA(f) (Met) and tRNA(m) (Met) isolated from either plus-folate or minus-folate cells. S. faecalis methionyl-tRNA formyltransferase displays optimal activity at pH values near 7.0, is stimulated by Mg(2+), and has an apparent molecular weight of approximately 29,900 when estimated by sucrose density gradient centrifugation. The K(m) value of plus-folate formyltransferase for plus-folate Met-tRNA(f) (Met) does not differ significantly from that of minus-folate formyltransferase for minus-folate Met-tRNA(f) (Met). Both enzymes can utilize either 10-formyltetrahydrofolate or 10-formyltetrahydropteroyltriglutamate as the formyl donor; the Michaelis constant for the monoglutamyl pteroyl coenzyme is slightly less than that of the triglutamyl pteroyl coenzyme for both transformylases. Tetrahydrofolate and uncharged tRNA(f) (Met) are competitive inhibitors of both plus- and minus-folate S. faecalis formyltransferase; folic acid, pteroic acid, aminopterin, and Met-tRNA(m) (Met) are not inhibitory. These results indicate that the presence or absence of folic acid in the culture medium of S. faecalis has no apparent effect on either methionyl-tRNA synthetase or methionyl-tRNA formyltransferase, the two enzymes directly involved in the formation of formylmethionyl-tRNA(f) (Met). Therefóre, the lack of N-formylation of Met-tRNA(f) (Met) in minus-folate S. faecalis is due to the absence of the formyl donor, a 10-formyl-tetrahydropteroyl derivative. Although the general properties of S. faecalis methionyl-tRNA synthetase are similar to those of other aminoacyl-tRNA synthetases, S. faecalis methionyl-tRNA formyltransferase differs from other previously described transformylases in certain kinetic parameters.     

Topographic modeling of free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer: bending of the 3'-terminal arm in tRNAfMet

Conformations of tRNAfMet, free and methionyl-tRNA synthetase bound forms, are analyzed by using singlet-singlet energy transfer as a spectroscopic ruler. tRNAfMet(8-13,3'-Flc), tRNAfMet(8-13,D-Etd), and tRNAfMet(3'-Flc,D-Etd) are prepared by sequential chemical modifications. The methionyl-tRNA synthetase binding affinity of these double-labeled tRNAfMets is similar to those of unmodified tRNAfMet. The fluorescence properties of the individual fluorophore in these tRNAs, including emission spectra, anisotropy, and quenching by methionyl-tRNA synthetase, are similar to those of single-labeled tRNAfMet. The transfer efficiencies of double-labeled tRNAfMets, as determined by both donor quenching and sensitized emission, showed efficient energy transfer in all cases. Random orientation being assumed, the apparent distances are 25 A between 8-13 and D20, 44 A between 8-13 and the 3'-terminus, and 49 A between the 3'-terminus and D20, respectively, in free tRNAfMet. Upon binding of methionyl-tRNA synthetase, the apparent distances are 25 A between 8-13 and D20, 45 A between 8-13 and the 3'-terminus, and 54 A between the 3'-terminus and D20, respectively. These results provide topographic models of these specific locations in free and methionyl-tRNA synthetase bound tRNAfMet and suggest that the immobilized 3'-terminal arm in the amino acid acceptor stem bends toward the inner loop of the L-shaped tRNA upon binding of methionyl-tRNA synthetase.     

Affinity chromatography on agarose-hexyl-adenosine-5'-phosphate of methionyl-tRNA synthetase from Escherichia coli. Application of the couplings between the methionine and ATP sites.

Recent studies by us [Biochemistry (1977) 16, 2570-2579] have shown that L-methioninol, a methionine analog lacking the carboxylate negative charge, enhances the affinity of AMP for methionyl-tRNA synthetase while L-methionine antagonizes the nucleotide binding. Such couplings between ligands of the enzyme have now been applied to affinity chromatography of methionyl-tRNA synthetase on an agarose-hexyl-adenosine-5'-phosphate gel (the spacer is attached to AMP at the adenine C-8 position). Retention of the enzyme on this gel column was shown to be dependent on the presence of appropriate concentrations of magnesium and of L-methioninol in the equilibration buffer. The enzyme was then specifically recovered from the column by omitting the amino alcohol or by adding an excess of L-methionine which antagonizes the cooperative effect of L-methioninol. This approach has provided the basis for a new purification procedure of methionyl-tRNA synthetase which leads to a 200-fold purification in a single chromatographic step. In this manner, after 30-50% ammonium sulfate fractionation of extracts of Escherichia coli EM 20031 (carrying the F32 episome), 0.25 mg X methionyl-tRNA synthetase was obtained at 90% purity per ml of agarose-hexyl-adenosine-5'-phosphate gel.     

Generation of multiple forms of methionyl-tRNA synthetase from the multi-enzyme complex of mammalian aminoacyl-tRNA synthetases by endogenous proteolysis

Methionyl-tRNA synthetase occurs free and as high-molecular-weight multi-enzyme complexes in rat liver. The free form is purified to near homogeneity by conventional column chromatography and affinity chromatography on tRNA-Sepharose. The native molecular weight of free methionyl-tRNA synthetase is 64 500, based on its sedimentation coefficient of 4.5 S and Stokes radius of 33 A. The free methionyl-tRNA synthetase apparently belongs to alpha-type subunit structure, since the subunit molecular weight is 68 000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Methionyl-tRNA synthetase is dissociated from the high-molecular-weight synthetase complex by controlled trypsinization, according to Kellermann, O., Viel, C. and Waller, J.P. (Eur. J. Biochem. 88 (1978) 197-204). The dissociated, free methionyl-tRNA synthetase is subsequently purified to near homogeneity. The subunit structure of dissociated methionyl-tRNA synthetase is identical to that of endogenous free methionyl-tRNA synthetase. Anti-serum raised against Mr 104 000 protein in the synthetase complex, specifically inhibited methionyl-tRNA synthetase in both the free and the high-molecular-weight forms to the same extent. These results suggest that the occurrence of multiple forms of methionyl-tRNA synthetases in mammalian cells may, in part, be due to proteolytic cleavage.     

Role of methionyl-transfer ribonucleic acid in the regulation of methionyl-transfer ribonucleic acid synthetase of Escherichia coli K-12.

A decrease in the in vivo acylation level of methionine transfer ribonucleic acid (tRNAmet) induced by methioninyl adenylate led to a specific derepression of methionyl-transfer ribonucleic acid (tRNA) synthetase formation. This derepression required de novo protein synthesis and was reflected by overproduction of unaltered enzyme. Two different strains of Escherichia coli K-12 that have normal levels of methionyl-tRNA synthetase were examined and the derepression of methionyl-tRNA synthetase was observed in both. Moreover, for one of these strains, the relation between the level of methionyl-tRNA synthetase and deacylation level of tRNAmet was established; under the growth conditions used, when more than 25% of tRNAmet was deacylated, methionyl-tRNA synthetase formation was derepressed and the level of derepression became proportional to the amount of tRNAmet deacylated. Concomitantly, the enzyme was subject to specific inactivation as a consequence of which the true de novo rate of derepression of the formation of this enzyme was higher than that determined by measurements of enzyme activity. These studies were extended to strains AB311 and ed2, which had a constitutive enhanced level of methionyl-tRNA synthetase. In these strains no derepression of enzyme formation was observed on reducing the acylation level of tRNAmet by use of methioninyl adenylate.