γ-Glutamylcysteine Synthetase from Proteus mirabilis

The distribution of γ-glutamylcysteine synthetase (l-glutamate: L-cysteine γ-ligase, EC 6.3.2.2) was investigated in bacteria, and the enzyme was purified from Proteus mirabilis approximately 9,000-fold with an over-all yield of 10%. The purification procedure included ammonium sulfate fractionation, protamine treatment, DEAE-cellulose and hydroxylapatite column chromatographies and Sephadex gel filtrations. The purified enzyme was homogeneous by the criteria of ultracentrifugation. It showed multiple bands on disc-polyacrylamide gel electrophoresis and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One band with a molecular weight of 62,000 was obtained on SDS-polyacrylamide gel electrophoresis after cross-linking of the enzyme with dimethylsuberimidate. The molecular weight was determined from the sedimentation and diffusion coefficients to be 64,000 and by Sephadex G-150 gel filtration to be 62,000. The purified enzyme catalyzed the stoichiometric formation of γ-glutamylcysteine and the reaction showed a sigmoidal dependence upon l-cysteine concentration. The enzyme also catalyzed γ-glutamyl amino acid formation from l-α-aminobutyrate, l-homoserine, glycine, l-serine, dl-norvaline or dl-homocysteine, but at lower rates than from l-cysteine. The γ-glutamyl-α-aminobutyrate formation by the enzyme did not show a sigmoidal but a hyperbolic dependence upon l-α-aminobutyrate concentration.     

Heavy Metal Tolerance and Accumulation in Indian Mustard (Brassica Juncea L.) Expressing Bacterial γ-Glutamylcysteine Synthetase or Glutathione Synthetase

The overexpression of either γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase (GS) in Brassica juncea transgenics was shown previously to result in higher accumulation of glutathione (GSH) and phytochelatins (PCs), as well as enhanced Cd tolerance and accumulation. The present study was aimed at analyzing the effects of γ-ECS or GS overexpression on tolerance to and accumulation of other metal/loids supplied individually in agar medium (seedlings) or in hydroponics (mature plants). Also, as pollution in nature generally consists of mixtures of metals, glutamylcysteine synthetase (ECS) and GS seedlings were tested on combinations of metals. Compared to wild-type plants, ECS and GS transgenics exhibited a significantly higher capacity to tolerate and accumulate a variety of metal/loids (particularly As, Cd, and Cr) as well as mixed-metal combinations (As, Cd, Zn/As, Pb, and Zn). This enhanced metal tolerance and accumulation of the ECS and GS transgenics may be attributable to enhanced production of PCs, sustained by a greater availability of GSH as substrate, as suggested by their higher concentrations of GSH, PC2, PC3, and PC4 as compared to wild-type plants. Overexpression of GS and γ-ECS may represent a promising strategy for the development of plants with an enhanced phytoremediation capacity for mixtures of metals.     

Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase

Pyrrolysyl-tRNA synthetase (PylRS), an aminoacyl-tRNA synthetase (aaRS) recently found in some methanogenic archaea and bacteria, recognizes an unusually large lysine derivative, l-pyrrolysine, as the substrate, and attaches it to the cognate tRNA (tRNA^Pyl). The PylRS-tRNA^Pyl pair interacts with none of the endogenous aaRS-tRNA pairs in Escherichia coli, and thus can be used as a novel aaRS-tRNA pair for genetic code expansion. The crystal structures of the Methanosarcina mazei PylRS revealed that it has a unique, large pocket for amino acid binding, and the wild type M. mazei PylRS recognizes the natural lysine derivative as well as many lysine analogs, including N^?-(tert-butoxycarbonyl)-l-lysine (Boc-lysine), with diverse side chain sizes and structures. Moreover, the PylRS only loosely recognizes the α-amino group of the substrate, whereas most aaRSs, including the structurally and genetically related phenylalanyl-tRNA synthetase (PheRS), strictly recognize the main chain groups of the substrate. We report here that wild type PylRS can recognize substrates with a variety of main-chain α-groups: α-hydroxyacid, non-α-amino-carboxylic acid, N_α-methyl-amino acid, and d-amino acid, each with the same side chain as that of Boc-lysine. In contrast, PheRS recognizes none of these amino acid analogs. By expressing the wild type PylRS and its cognate tRNA^Pyl in E. coli in the presence of the α-hydroxyacid analog of Boc-lysine (Boc-LysOH), the amber codon (UAG) was recoded successfully as Boc-LysOH, and thus an ester bond was site-specifically incorporated into a protein molecule. This PylRS-tRNA^Pyl pair is expected to expand the backbone diversity of protein molecules produced by both in vivo and in vitro ribosomal translation.     

Synthesis of γ-Glutamylpeptides by γ-Glutamylcysteine Synthetase from Proteus mirabilis

Syntheses of various γ-glutamylpeptides were examined taking use of the highly purified γ-glutamylcysteine synthetase from Proteus mirabilis. The accumulation of each peptide was measured after long time incubation, and good formation was observed in the synthesis of peptides of following amino acids, l-cysteine, l-α-aminobutyrate, l-serine, l-homoserine, glycine, l-alanine, l-norvaline, l-lysine, l-threonine, taurine and l-valine. Peptide syntheses were confirmed by analyses of the component amino acids, after hydrolysis of the peptides.

The structure of the glutamylpeptides, especially the peptide-linkage at the γ-carbonyl residue of l-glutamate, was determined by mass spectrometry of the N-trifluoroacetyl methylester derivatives of the glutamylpeptides. Enzymatic synthesis of γ-glutamyl-l-α-aminobutyrate was also confirmed by PMR spectrometry in the comparison with chemically synthesized compound.     

Purification and Characterization of γ-Glutamylmethylamide Synthetase from Methylophaga sp. AA-30

γ-Glutamylmethylamide synthetase [L-glutamate: methylamine ligase (ADP-forming), EC 6.3.4.12] was purified about 70-fold from a cell-free extract of Methylophaga sp. AA-30 by ammonium sulfate fractionation, Octyl-Sepharose column chromatography, and Sephacryl S-300 gel filtration. Only a single protein band was detected after SDS-polyacrylamide gel electrophoresis of the purified preparation; the band was at a position corresponding to a molecular weight of 56,000. The molecular weight of the enzyme was calculated to be 440,000 by Superose 6HR gel filtration, so we suggest that the enzyme is an octomer of identical subunits. The enzyme had maximum activity at pH 7.5 and 40°C. It could use ethylamine and propylamine instead of methylamine as the substrate, but it could not use D-glutamate or L-glutamine instead of L-glutamate.     

Occurrence of Isóenzymes of Glutamine Synthetase in the Alga Chlorella kessleri

Two forms (GS₁ and GS₂) of glutamine synthetase have been isolated, separated by ion exchange chromatography, and partly characterized from cells of the green alga Chlorella kessleri. Both forms are present in cells grown autotrophically or heterotrophically on various nitrogen sources, but under all nutritional conditions GS₁ was found to be the major isoenzyme present (60-80%). The activity of both isoenzymes was greatest in cells grown under nitrogen-limiting conditions. Both isoenzymes have molecular weights in the range 340 to 350,000 daltons. GS₁ was found to have a greater thermostability than GS₂: GS₁ was stable at 30°C while GS₂ lost 95% of its activity in 30 minutes. GS₁ was much less sensitive to thiol reactive reagents than GS₂.     

Cell-Type-Specific Activation of the Oligoadenylate Synthetase–RNase L Pathway by a Murine Coronavirus

Previous studies have demonstrated that the murine coronavirus mouse hepatitis virus (MHV) nonstructural protein 2 (ns2) is a 2′,5′-phosphodiesterase that inhibits activation of the interferon-induced oligoadenylate synthetase (OAS)-RNase L pathway. Enzymatically active ns2 is required for efficient MHV replication in macrophages, as well as for the induction of hepatitis in C57BL/6 mice. In contrast, following intranasal or intracranial inoculation, efficient replication of MHV in the brain is not dependent on an enzymatically active ns2. The replication of wild-type MHV strain A59 (A59) and a mutant with an inactive phosphodiesterase (ns2-H126R) was assessed in primary hepatocytes and primary central nervous system (CNS) cell types—neurons, astrocytes, and oligodendrocytes. A59 and ns2-H126R replicated with similar kinetics in all cell types tested, except macrophages and microglia. RNase L activity, as assessed by rRNA cleavage, was induced by ns2-H126R, but not by A59, and only in macrophages and microglia. Activation of RNase L correlated with the induction of type I interferon and the consequent high levels of OAS mRNA induced in these cell types. Pretreatment of nonmyeloid cells with interferon restricted A59 and ns2-H126R to the same extent and failed to activate RNase L following infection, despite induction of OAS expression. However, rRNA degradation was induced by treatment of astrocytes or oligodendrocytes with poly(I·C). Thus, RNase L activation during MHV infection is cell type specific and correlates with relatively high levels of expression of OAS genes, which are necessary but not sufficient for induction of an effective RNase L antiviral response.     

Serum Glutamine Synthetase Has No Value as a Diagnostic Biomarker for Alzheimer’s Disease

In order to test whether serum glutamine synthetase (GS) is of potential diagnostic value for Alzheimer’s disease (AD), we set up a study to compare serum GS concentrations between AD patients and control subjects. The study population (n = 165) consisted of AD patients (n = 94) and age-matched (n = 41) and age-unmatched (n = 30) control subjects. Serum GS analysis was performed by means of ELISA. No significant differences in serum GS levels were found between the AD group and age-matched controls. Age correlated positively with serum GS concentrations in AD patients and control subjects. This study suggests that serum GS levels have no diagnostic value for AD.     

Characterization of a Bifunctional Cytidine 5′-Monophosphate N-acetylneuraminic acid Synthetase Cloned from Streptococcus Agalactiae

Recombinant CMP-sialic acid synthetase, cloned from Streptococcus agalactiae serotype V strain 2603 V/R, is bifunctional having both CMP-sialic acid synthetase and acetylhydrolase (acylesterase) activities. The enzyme is active over a wide pH range with an optimal CMP-sialic acid synthetase activity at pH 9.0 and an optimal acetylhydrolase activity at pH 8.0. A metal cofactor (either Mg²⁺ or Mn²⁺) is required for the CMP-sialic acid synthetase activity but is not for acetylhydrolase activity. Both catalytic functions, however, are impaired by high concentrations of Mn²⁺. Received 10 August 2005; Revisions requested 30 August 2005; Revisions received 1 November 2005; Accepted 2 November 2005     

γ-Glutamylcysteine Production by Escherichia coli B Cells Dosed with the γ-Glutamylcysteine Synthetase Gene

Molecular studies on the evolution and systematics of fungi have been established primarily based on the neutral theory by analyzing neutral mutations in some defined segments of housekeeping genes as genetic markers. Such an approach is, however, hardly applicable for analyzing ancient evolutionary radiations. In the present study, we looked for DNA sequences characterizing higher taxa, and discovered a unique macroevolutionary genomic marker, megB1, that specifies the phylum Basidiomycota. megB1 is an approximately 500-bp DNA element, which is defined by terminal sequences and five internal segments conserved throughout the phylum. megB1 resides on the rDNA intergenic spacer 1 (IGS1) from 27 species of 10 Basidiomycota genera examined. While megB1 was not found in IGS1 from the other 92 species of the 27 Basidiomycota genera, several genera representing them carry megB1 in some other genomic regions. No known taxonomic criteria fit into the classification on the basis of whether megB1 resides on rDNA. Neighbor-joining analysis of the megB1 sequence, however, properly assigned species to their respective genera. Thus far, megB1 has not been found in any genomic or genetic databases currently available for other phyla. These results suggest that megB1 may have emerged upon the occurrence of Basidiomycota, and that this phylum evolved thereafter leaving this element conserved throughout their further differentiation. megB1 may be a novel genomic marker useful in the analysis of ancient through the latest evolutionary radiation in Basidiomycota.     

Estrogen (17β-Estradiol) Enhances Glutamine Synthetase Activity in C6-Glioma Cells

Glutamine synthetase (GS) is the major glutamine-forming enzyme of vertebrates and is accepted to be a marker of astroglial cells. Maturation of astroglial cells is characterized by an increase of GS activity, and the regulation of this enzyme is the topic of many publications. Because of the fundamental role of the GS in controlling brain glutamate and glutamine level, it is essential to understand the mechanism of expression of this enzyme. To our knowledge, the effect of estrogen (17β-estradiol) on GS activity in glial cells has not been reported. We examined the effect of treatment with estrogen on glutamine synthetase enzyme activity in glial cells. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well-established model system. We adapted a colorimetric method to measure GS-catalyzed γ-glutamyltransferase (GT) activity in C6-glioma cells. The assay monitors GT activity of glutamine synthetase by following the absorbance of the product γ-glutamyl hydroxamate at 540 nm. We observed that, the absorbance of γ-glutamyl hydroxamate significantly increased in estrogen treated cells (0.13±0.03), as compared to untreated cells (0.058±0.015). Estrogen also significantly increased concentration of glutamine in C6-glioma cells as measured by fluorometric assay. In addition, western blot analysis showed that estrogen significantly increased the amount of glutamine synthetase compared to control. This estrogen effect could have important physiological implications on cerebral glutamate and glutamine metabolism.     

Glutamine Synthetase Stability and Subcellular Distribution in Astrocytes Are Regulated by γ-Aminobutyric Type B Receptors

Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABA_BRs) are expressed by astrocytes within the mammalian brain. GABA_BRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABA_BR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABA_BR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABA_BR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABA_BRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABA_BRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.     

A master switch couples Mg²⁺-assisted catalysis to domain motion in B. stearothermophilus tryptophanyl-tRNA Synthetase

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Highlights? Combinatorial mutagenesis at four remote sites shifts reaction coordinate stabilities ? Cooperative action of all four sites accounts for the entire catalytic role of Mg2⁺ ? Catalysis of ATP utilization by Mg2⁺ occurs if, and only if, the conformation changes ? This type of allosteric effect may explain vectorial coupling in transducing NTPases     

ε-Poly-l-Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly-l-Lysine Synthetase

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.     

Acyl Coenzyme A Synthetase from Pseudomonas fragi Catalyzes the Synthesis of Adenosine 5′-Polyphosphates and Dinucleoside Polyphosphates

Acyl coenzyme A (CoA) synthetase (EC 6.2.1.8) from Pseudomonas fragi catalyzes the synthesis of adenosine 5′-tetraphosphate (p₄A) and adenosine 5′-pentaphosphate (p₅A) from ATP and tri- or tetrapolyphosphate, respectively. dATP, adenosine-5′-O-[γ-thiotriphosphate] (ATPγS), adenosine(5′)tetraphospho(5′)adenosine (Ap₄A), and adenosine(5′)pentaphospho(5′)adenosine (Ap₅A) are also substrates of the reaction yielding p₄(d)A in the presence of tripolyphosphate (P₃). UTP, CTP, and AMP are not substrates of the reaction. The K_m values for ATP and P₃ are 0.015 and 1.3 mM, respectively. Maximum velocity was obtained in the presence of MgCl₂ or CoCl₂ equimolecular with the sum of ATP and P₃. The relative rates of synthesis of p₄A with divalent cations were Mg = Co > Mn = Zn >> Ca. In the pH range used, maximum and minimum activities were measured at pH values of 5.5 and 8.2, respectively; the opposite was observed for the synthesis of palmitoyl-CoA, with maximum activity in the alkaline range. The relative rates of synthesis of palmitoyl-CoA and p₄A are around 10 (at pH 5.5) and around 200 (at pH 8.2). The synthesis of p₄A is inhibited by CoA, and the inhibitory effect of CoA can be counteracted by fatty acids. To a lesser extent, the enzyme catalyzes the synthesis also of Ap₄A (from ATP), Ap₅A (from p₄A), and adenosine(5′)tetraphospho(5′)nucleoside (Ap₄N) from adequate adenylyl donors (ATP, ATPγS, or octanoyl-AMP) and adequate adenylyl acceptors (nucleoside triphosphates).Dinucleoside polyphosphates have been detected in a wide variety of eukaryotic and prokaryotic organisms (13). In higher organisms, their concentrations are generally on the order of 0.01 to 1 μM. Human blood platelets and chromaffin cells of bovine adrenal medulla contain diadenosine polyphosphates located in the dense bodies (10, 26, 35) and chromaffin granules (32, 38), respectively, where they may reach higher local concentrations. The occurrence of dinucleoside polyphosphates has been described for lower eukaryotic (Saccharomyces cerevisiae, Dictyostelium discoideum, and Physarum polycephalum) and for prokaryotic (Salmonella typhimurium, Escherichia coli, and Clostridium acetobutylicum) organisms (13).Dinucleoside tetraphosphates participate in the control of purine nucleotide metabolism (36), where Ap₄A is an activator of both the IMP-GMP-specific cytosolic 5′-nucleotidase (EC 3.1.3.5) and AMP deaminase (EC 3.5.4.6) (K_a, micromolar range) and Gp₄G is an activator of GMP reductase (EC 1.6.6.8) (K_a, nanomolar range) (36). As the concentration of dinucleoside polyphosphates increases under unfavorable environmental conditions, they have been implicated in the cellular response to stress (31). A role of Ap₄A in DNA synthesis has been proposed elsewhere (14). Dinucleoside polyphosphates are also transition state analogs of some kinases (37). More recently, the dinucleoside triphosphatase activity of a putative tumor suppressor gene product has been described (3).The nucleoside 5′-polyphosphates (p_nN) are another family of related compounds, p₄A has been detected in rabbit and horse muscle (41), rat liver (44), S. cerevisiae spores (19), and chromaffin granules (38). As p₄A is a very strong inhibitor (K_i, nanomolar range) of asymmetrical dinucleoside tetraphosphatase (EC 3.6.1.17) (22), changes in the level of p₄A could affect the concentration and physiological roles of Ap₄A. Other enzymes known to be inhibited (K_i, micromolar range) by p₄N are guanylate cyclase (EC 4.6.1.2) (p₄A and p₄G) (18) and phosphodiesterase I (EC 3.1.4.1) (p₄G) (9). Effects of p₄A on the tone of the vascular system, mediated by P₂ receptors, have also been described elsewhere (21).The cellular level of dinucleoside polyphosphates results from their rate of degradation and synthesis. The following specific enzymes, implicated in the cleavage of dinucleoside polyphosphates, have been described (see reference 15 for a review): asymmetrical dinucleoside tetraphosphatase (EC 3.6.1.17), symmetrical dinucleoside tetraphosphatase (EC 3.6.1.41), dinucleoside tetraphosphate phosphorylase (EC 2.7.7.53), and dinucleoside triphosphatase (EC 3.6.1.29). In addition, there are other unspecific enzymes able to catalyze the hydrolysis of dinucleoside polyphosphates like E. coli 5′-nucleotidase (34) and phosphodiesterase I (9, 15, 26).This paper deals with the synthesis of (di)nucleoside polyphosphates. It has been known since 1966 that some aminoacyl tRNA synthetases (30, 45) catalyze the synthesis of Ap₄A through reactions 1 and 2: reaction 1 reaction 2 The possibility that other enzymes (mainly synthetases and some transferases) which catalyze the formation of AMP, via nucleotidyl-containing intermediates and by releasing PP_i, could catalyze the synthesis of dinucleoside polyphosphates was later raised (17). Luciferase (EC 1.13.12.7), considered as an oxidoreductase, catalyzes the synthesis of Ap₄A with ATP as substrate and luciferin as an essential activator (27, 40): reaction 3 reaction 4 Acetyl-CoA synthetase (EC 6.2.1.1) from S. cerevisiae also catalyzes the synthesis of p₄A and p₅A, from ATP and P₃ and P₄, respectively (16). In the reactions catalyzed by luciferase and acetyl-CoA synthetase, ATP is a very good substrate for the formation of the E · X-AMP complex (X = the appropriate acyl residue), whereas any NTP (or even P₃) is an acceptor (particularly in the case of luciferase) of the AMP moiety of the complex, provided that it has an intact terminal pyrophosphate (27, 40).Here we show that acyl-CoA synthetase from Pseudomonas fragi catalyzes the synthesis of p₄A, p₅A, Ap₄A, Ap₅A, and a variety of Ap₄Ns. In our view, these findings widen the knowledge of the mechanisms of synthesis of (di)nucleoside polyphosphates in prokaryotes and, by extrapolation, also in eukaryotes.     

Biosynthesis of Albomycin δ(2) Provides a Template for Assembling Siderophore and Aminoacyl-tRNA Synthetase Inhibitor Conjugates

"Trojan horse" antibiotic albomycins are peptidyl nucleosides consisting of a highly modified 4'-thiofuranosyl cytosine moiety and a ferrichrome siderophore that are linked by a peptide bond via a serine residue. While the latter component serves to sequester iron from the environment, the seryl nucleoside portion is a potent inhibitor of bacterial seryl-tRNA synthetases, resulting in broad-spectrum antimicrobial activities of albomycin δ(2). The isolation of albomycins has revealed this biological activity is optimized only following two unusual cytosine modifications, N4-carbamoylation and N3-methylation. We identified a genetic locus (named abm) for albomycin production in Streptomyces sp. ATCC 700974. Gene deletion and complementation experiments along with bioinformatic analysis suggested 18 genes are responsible for albomycin biosynthesis and resistance, allowing us to propose a potential biosynthetic pathway for installing the novel chemical features. The gene abmI, encoding a putative methyltransferase, was functionally assigned in vitro and shown to modify the N3 of a variety of cytosine-containing nucleosides and antibiotics such as blasticidin S. Furthermore, a ΔabmI mutant was shown to produce the descarbamoyl-desmethyl albomycin analogue, supporting that the N3-methylation occurs before the N4-carbamoylation in the biosynthesis of albomycin δ(2). The combined genetic information was utilized to identify an abm-related locus (named ctj) from the draft genome of Streptomyces sp. C. Cross-complementation experiments and in vitro studies with CtjF, the AbmI homologue, suggest the production of a similar 4'-thiofuranosyl cytosine in this organism. In total, the genetic and biochemical data provide a biosynthetic template for assembling siderophore-inhibitor conjugates and modifying the albomycin scaffold to generate new derivatives.     

Biallelic Mutations of Methionyl-tRNA Synthetase Cause a Specific Type of Pulmonary Alveolar Proteinosis Prevalent on Réunion Island

Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to tRNA and is critical for protein biosynthesis. We identified biallelic missense mutations in MARS in a specific form of pediatric pulmonary alveolar proteinosis (PAP), a severe lung disorder that is prevalent on the island of Réunion and the molecular basis of which is unresolved. Mutations were found in 26 individuals from Réunion and nearby islands and in two families from other countries. Functional consequences of the mutated alleles were assessed by growth of wild-type and mutant strains and methionine-incorporation assays in yeast. Enzyme activity was attenuated in a liquid medium without methionine but could be restored by methionine supplementation. In summary, identification of a founder mutation in MARS led to the molecular definition of a specific type of PAP and will enable carrier screening in the affected community and possibly open new treatment opportunities.     

Nα-Ipteroyltetra (γ-Glutamyl)]-Lysine as a Ligand for the Purification of Thymidylate Synthetase by Affinity Chromatography

N^α[Pteroyltetra (γ-glutamyl)]-lysine Sepharose was synthesized and shown to be a stable, high capacity affinity matrix capable of bringing about the purification of Lactobacillus casei thymidylate synthetase to maximum specific activity from crude extracts in high yield. Under conditions optimal for binding of thymidylate synthetase, dihydrofolate reductase was not bound.