Large-Scale and Small-Scale Methods for the Preparation of 5,10-Methylenetetrahydrofolate

Abstract

Two procedures have been developed for the synthesis and isolation of 5,10-methylenetetrahydrofolate, the cofactor for the reaction catalyzed by thymidylate synthesize, one of which can be used for large-scale preparations of the cofactor and the other for small-scale syntheses especially suitable for obtaining the radio labeled cofactor. The large-scale procedure involves treatment of folic acid with dithionite to give dihydrofolate, which is then converted to tetrahydrofolate by dihydrofolate reductase (L. casei). The small-scale method involves a direct enzymatic reduction of folic acid to tetrahydrofolate by dihydrofolate reductase, and has been used to prepare the double-labeled 5,10-[¹⁴C]methylene[3′,5′,7,9-³H]tetrahydrofolate. In both procedures, after the reduction steps have been performed, the tetrahydrofolate is treated in situ with formaldehyde prior to purification by DEAE-cellulose chromatography, thus allowing the isolation of 5,10-methylenetetrahydrofolate as a dry powder after lyophilization. This product is active in the enzyme reaction without the further addition of excess formaldehyde as in previous procedures. The cofactor prepared in this manner has much improved stability toward oxidation compared to free tetrahydrofolate.     

Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase

Tritiated forms of polyglutamyl folates are not commercially available but are often needed for experimental uses in folate biochemistry. Thus, considerable interest exists in the preparation of polyglutamyl [³H]folates from the commercial monoglutamyl [³H]folates. However, refinement of established enzymatic and biological synthesis methods is still needed. To address this need we developed improved procedures for the conversion of monoglutamyl [³H]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of 1 ng/ml (2.27 nM) [³H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10 mM β-mercaptoethanol and heated to release the folates. The extracted [³H]folates were purified on a folate-binding protein affinity column and then applied to a Sephadex G-10 column to separate the eluted poly- from the monoglutamyl folate species. High performance liquid chromatography with multichannel electrochemical detection indicated that the bacterial synthesis yielded predominantly polyglutamates of [³H]5-methyltetrahydrofolate and [³H]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [³H]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [³H]folate substrate.     

Identification of folate binding macromolecule in rabbit choroid plexus.

A macromolecular binder of folic acid and folic acid derivatives has been identified in the particulate fraction of homogenates of rabbit choroid plexus. Within the choroid plexus, there are 2.3 nmol of folate-binding activity (binder) per g of tissue. The molecular weight of the folate binder complex, separated from the particulate fraction after solubilization with Triton X-100, was 340,000 to 400,000 by Sephadex gel filtration. The partially purified binder, when freed of endogenous folates, bound equivalent amounts of both [3H]folic acid and [methyl-14C]methyltetrahydrofolic acid per mg of protein. Folic acid, homofolic acid, 5-methyltetrahydrofolic acid, and to a lesser degree, methotrexate, inhibited the binding of both [3H]folic acid and [14C]methyltetrahydrofolic acid. Binding activity, which decreased below pH = 7.0, was unaffected by pretreatment with ribonuclease but was eliminated completely by papain and a protease (Streptomyces griseus). Although dihydrofolate reductase was present in choroid plexus, the binder was distinct from dihydrofolate reductase as judged by gel filtration and methotrexate sensitivity. This high affinity binder of folates may be responsible, in part, for the rapid, saturable uptake of folic acid and methyltetrahydrofolic acid by rabbit choroid plexus in vitro.     

A flow microcalorimetric method for enzyme activity measurements: Application to dihydrofolate reductase

A flow microcalorimetric method was developed for the analysis of enzymatic activities in crude tissue homogenates. It can be applied whenever a heat exchange is involved in an enzymatic reaction. The consequent sensitivity obviously depends on the enthalpy variation observed. Dihydrofolate reductase was chosen as an example; this enzyme is the molecular target of methotrexate, a widely used anticancer agent. This calorimetric method, whose sensitivity limit is 1.48 × 10⁻⁴ units of dihydrofolate reductase per milliliter of reactant medium, allows enzyme activity measurements in tissues with low dihydrofolate reductase levels. A few examples of measurements in animal tissues are given. These measurements are of some interest; indeed, increased activity and increased levels of this enzyme are two of the mechanisms which may explain resistance to methotrexate.     

Folate metabolism in the superior cervical ganglion histochemical study

Summary By means of histochemical methods, folic acid, dihydrofolate reductase and NADH₂-cytochrome-C-reductase were studied in the bovine superior cervical ganglion, in parallel with quantitative estimations of dihydrofolate reductase activity and in connection with the process of ageing. Various levels of folate metabolism were present in nerve cells and glial cells, as well as in pre or postganglionic nerves. In the process of ageing the activity of dihydrofolate reductase gradually decreased and the folic acid concentration in the nerve cells increased. Thus the enzyme — substrate ratio appeared to favour the enzyme in young animals but the substrate in old animals.     

Nutritional Requirements of Plasmodium falciparum in Culture. III. Further Observations on Essential Nutrients and Antimetabolites1

In a semi-defined minimal medium for cultivation of Plasmodium falciparum, ribose, mannose, fructose, galactose, and maltose could not replace glucose. Hypoxanthine was the preferred purine source for the parasite over adenine, guanine, inosine, adenosine and guanosine although all supported growth equally. Inhibitors of nucleoside uptake had low potency in killing the parasites but depressed incorporation of [³H]adenosine more than [³H]hypoxanthine. Glutamate could not be replaced by 5-oxoproline, indicating that the γ-glutamyl transferase pathway for amino acid uptake is probably not found in this organism. Adenine, nicotinamide, and orotic acid could not supplement glutamine-deficient medium. The pyridoxine antagonists isoniazid and 4-deoxypyridoxine were reversed by amino acid supplementation, suggesting that transaminases may be targets of these drugs. Orotic acid, but not glutathione or its amino acid components, partially reversed the effects of 8-methylamino-8-desmethyl riboflavin. Thus, the flavin enzyme, dihydroorotic acid dehydrogenase, but not glutathione reductase, appears to be a target of this riboflavin antagonist. Five biotin antagonists had no significant activity. The choline antagonist 2-(tert-butylamino)ethanol and thiamin uptake inhibitors had nonspecific inhibitory effects, which were not reversed by the respective target vitamin. Buthionine sulfoximine and methionine sulfoximine, inhibitors of glutathione synthesis, had significant oxygen-dependent toxicity. Six sulfonamides showed marked variation in potency and efficacy. Sulfathiazole and sulfadoxine were reversed differentially by p-aminobenzoic acid, folic acid, and folinic acid. Folinic acid was more effective than folic acid at reversing the toxicity of the dihydrofolate reductase inhibitors aminopterin and pyrimethamine; p-aminobenzoic acid had no effect.     

Effects of 5-fluorouracil on dihydrofolate reductase and dihydrofolate reductase mRNA from methotrexate-resistant KB cells

Growth of methotrexate-resistant dihydrofolate reductase gene-amplified KB cells in the presence of 5-fluorouracil results in an increase in dihydrofolate reductase mRNA. This increase can be solely attributed to a species of RNA of approximately 3.5 kilobase pairs in size. Although dihydrofolate reductase enzyme activity increases per cell with increasing 5-fluorouracil, there is a decrease of enzyme activity per mg of protein (Dolnick, B. J., and Pink, J. J. (1983) J. Biol. Chem. 258, 13299-13306). The rate of in vivo enzyme synthesis, as assayed by immunoprecipitation and supported by gel electrophoresis, does not decrease and may in fact increase with increasing 5-fluorouracil. Translation of purified dihydrofolate reductase mRNA in vitro shows that the rate of translation is unaffected by 5-fluorouracil incorporation into mRNA. The inhibition of dihydrofolate reductase by a monospecific polyclonal antiserum is reduced with extracts from 5-fluorouracil-treated cells. Inhibition of dihydrofolate reductase by methotrexate is significantly reduced in extracts from 5-fluorouracil-treated cells compared to control extracts. Tight binding of [3H]methotrexate is also different in extracts from 5-fluorouracil-treated cells. This data supports the hypothesis of translational miscoding during protein synthesis as a major mechanism of 5-fluorouracil-mediated cytotoxicity and suggests a new mechanism of 5-fluorouracil-methotrexate antagonism.     

Subcellular localization of γ-glutamyl carboxypeptidase and of folates

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [³H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions.The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85–90% folate polyglutamates.Injected [³H] folic acid was initially concentrated in the microsomal fraction, as measured by ³H cpm per ng folate.Initially, injected [³H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total ³H cpm determined to be [³H] folate penta- and hexaglutamates. At 24 h, the conversion of [³H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates.Injected [³H] folic acid after 2 h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.     

Folate metabolism in the superior cervical ganglion histochemical study.

By means of histochemical methods, folic acid, dihydrofolate reductase and NADH2-cytochrome-C-reductase were studied in the bovine superior cervical ganglion, in parallel with quantitative estimations of dihydrofolate reductase activity and in connection with the process of ageing. Various levels of folate metabolism were present in nerve cells and glial cells, as well as in pre or postganglionic nerves. In the process of ageing the activity of dihydrofolate reductase gradually decreased and the folic acid concentration in the nerve cells increased. Thus the enzyme --- substrate ratio appeared to favour the enzyme in young animals but the substrate in old animals.     

Solvent environments significantly affect the enzymatic function of Escherichia coli dihydrofolate reductase: Comparison of wild-type protein and active-site mutant D27E

To investigate the contribution of solvent environments to the enzymatic function of Escherichia coli dihydrofolate reductase (DHFR), the salt-, pH-, and pressure-dependence of the enzymatic function of the wild-type protein were compared with those of the active-site mutant D27E in relation to their structure and stability. The salt concentration-dependence of enzymatic activity indicated that inorganic cations bound to and inhibited the activity of wild-type DHFR at neutral pH. The BaCl₂ concentration-dependence of the ¹H–¹⁵N HSQC spectra of the wild-type DHFR–folate binary complex showed that the cation-binding site was located adjacent to the Met20 loop. The insensitivity of the D27E mutant to univalent cations, the decreased optimal pH for its enzymatic activity, and the increased K_m and K_d values for its substrate dihydrofolate suggested that the substrate-binding cleft of the mutant was slightly opened to expose the active-site side chain to the solvent. The marginally increased fluorescence intensity and decreased volume change due to unfolding of the mutant also supported this structural change or the modified cavity and hydration. Surprisingly, the enzymatic activity of the mutant increased with pressurization up to 250 MPa together with negative activation volumes of ? 4.0 or ? 4.8 mL/mol, depending on the solvent system, while that of the wild-type was decreased and had positive activation volumes of 6.1 or 7.7 mL/mol. These results clearly indicate that the insertion of a single methylene at the active site could substantially change the enzymatic reaction mechanism of DHFR, and solvent environments play important roles in the function of this enzyme.     

Trimethoprim resistance plasmids of different origin encode different drug-resistant dihydrofolate reductases.

Several plasmids mediating resistance to folic acid analogs were studied. The plasmids were in part newly isolated from clinical material and in part R factors studied earlier, such as R483, R721, R751, and R388. By gel chromatography, plasmid-carrying bacterial strains were all found to produce drug-resistant dihydrofolate reductases of a molecular weight distinctly larger than that of the chromosomal enzyme of the host. By gel electrophoresis and zymographic detection technique, analog inhibition characteristics, heat sensitivity, and pH optimum curves, the dihydrofolate reductases induced by R483, R751, and R388, respectively, could be clearly discerned as separate enzymes. Of the newly isolated plasmids all but one coded for a dihydrofolate reductase similar to that of R483. The aberrant one seemed to yield a new enzyme variant as judged from its drug inhibition characteristics and its pH optimum profile. Large differences in drug insensitivity were observed, thus the R751 and R388 enzymes were virtually insensitive to folic acid analogs, whereas the corresponding enzymes from the newly isolated plasmids, and from R483 showed a substantially higher sensitivity. On the other hand these latter enzymes were overproduced, in that the plasmid-carrying bacteria showed a 10- to 20-fold higher content of dihydrofolate reductase than the plasmid-free host strain. Among newly isolated trimethoprim-resistant strains, one was found which overproduced dihydrofolate reductase about 200-fold. In this case the enzyme was only slightly more resistant to folic acid analogs than the chromosomal Escherichia coli K-12 enzyme, and did not seem to be plasmid borne.     

Synthesis and incorporation of [6,7]-selenatryptophan into dihydrofolate reductase

Until recently, the only selenium containing amino acid which could be used to completely substitute for a wild type amino acid was selenomethionine (SeMet). In the last decade the preparation of SeMet containing proteins has proved to be valuable tools in the determination of three-dimensional structure by multiwavelength anomalous diffraction (MAD) techniques. The potential utility of a selenium containing tryptophan analog, beta-seleno[3,2-b]pyrrolyl-L-alanine ([4,5]selenatryptophan), has recently been demonstrated in the literature. This finding shows promise for the bioincorporation of its positional isomer, beta-selenolo[2,3-b]pyrrolyl-L-alanine ([6,7]selenatryptophan), thereby adding to the essential arsenal of selenium-containing amino acids for use in the characterization of proteins. The synthesis of [6,7]selenatryptophan by enzymatic biotransformation with tryptophan synthase from selenolo[2,3-b]pyrrole was carried out as well as its characterization by NMR spectroscopy and thin layer chromatography. Selenatryptophyl dihydrofolate reductase ([6,7]SeTrp-DHFR) was then synthesized in vivo, purified, and found to exhibit no perturbations to enzymatic activity.     

A radiometric assay for 5-enolpyruvylshikimate-3-phosphate synthase

A new assay for 5-enolpyruvylshikimate-3-phosphate synthase is described. This enzyme of the shikimate pathway of aromatic amino acid biosynthesis generates 5-enolpyruvylshikimate 3-phosphate and orthophosphate from phosphoenolpyruvate and shikimate 3-phosphate. The shikimate pathway is present in bacteria and plants but not in mammals. The assay employs a paper-chromatographic separation of radiolabeled substrate from product. The method is specific, is sensitive to 50 pmol of product, and is suitable for use in crude extracts of bacteria. This enzyme appears to be the primary target site of the commercial herbicide glyphosate (N-phosphonomethyl glycine). A procedure for the enzymatic synthesis of [¹⁴C]shikimate 3-phosphate from the commercially available precursor [¹⁴C]shikimic acid is also described.     

Turnover of dihydrofolate reductase in rapidly dividing cells

The turnover of dihydrofolate reductase has been studied in rapidly dividing cells of mouse lymphoma L1210 and Lactobacillus casei. Cells in culture were exposed to [¹⁴C]leucine for 24 hr and the subsequent decrease in radioactivity of the enzyme was followed as a function of time. The L1210 enzyme was isolated in pure form by subjecting the cell sonicate to affinity chromatography on amethopterin-Sepharose. The L. casei cells were processed by a multistep procedure which yielded the pure enzyme in both of its principal forms: (I), without TPNH; and (II), containing an equimolal amount of noncovalently bound TPNH. The half-lives ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2></mtext>}}$) of dihydrofolate reductase in the amethopterin-sensitive L1210 cells (L1210/S) and in the cells of a partially resistant subline (L1210/R2), characterized by an 8-fold increase in enzyme level, were 18 and 19 hr. When these cells were grown in the presence of sublethal concentrations of amethopterin, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values were increased to 39 and 90 hr. These results suggest that the transient increase in dihydrofolate reductase activity, observed when cells are exposed to amethopterin, is due largely to a decreased susceptibility of the enzyme-inhibitor complex to degradation. Bound TPNH also increases the half-life of dihydrofolate reductase as shown by the fact that forms (I) and (II) of the L. casei enzyme had $\text{t}{}_{\mathrm{<mtext>x1</mtext><mtext>2</mtext>}}$ values of approximately 3 and 9 hr.     

13C NMR evidence of the slow exchange of tryptophans in dihydrofolate reductase between stable conformations.

¹³C NMR spectra are reported for dihydrofolate reductase of $\text{Streptococcus}\text{faecium}$ labeled with [γ-¹³C]tryptophan. Two of the four tryptophans generate unusual resonances indicating slow exchange of the residues between alternative stable conformations. Since 3′, 5′-dichloromethotrexate sharpens two of the resonances, it apparently locks the corresponding residues into one conformation.     

A rapid radiometric assay for dihydrofolate reductase

A rapid radiochemical procedure for the measurement of dihydrofolate reductase activity is described. The method employs separation of the radiolabeled substrate from the products of the reaction by precipitation with acetic acid and zinc sulfate. This method permits rapid processing of samples and climinates time-consumlng column chromatography techniques. Good agreement of results is obtained when the radioactive method is compared to the spectrophotometric assay method.     

The occurrence and properties of dihydrofolate reductase in pea seedlings

Dihydrofolate reductase (E.C. 1.5.1.3 [EC] ) was found in pea seedlingsand was partially purified by treatments with ammonium sulfate,protamine sulfate and by DEAE-cellulose column chromatography.Some properties of the enzyme were investigated. Optimum pHfor the reaction was 6.5. In the enzyme reaction, FAH₂ and NADPH₂were specifically required. MICHAELIS constants for FAH₂ andNADPH₂ were 4.3x10^–6 M and 4.0x10^–5 M, respectively.Folate antagonists such as aminopterin, methotrexate and pyrimethaminewere potent inhibitors of this enzyme. Enzyme activity was almostcompletely inhibited at a concentration of 10^–7 M of aminopterinand methotrexate and 10^–6 M of pyrimethamine. Growth of germinating pea seeds was inhibited by aminopterin,methotrexate and pyrimethamine, and it recovered significantlywith a tetrahydro-derivative of folate, CF, but not with dihydrofolicor folic acid. These results suggest that growth inhibitionof pea seedlings by these antagonists is due to inhibition ofdihydrofolate reductase in seedlings. ¹Studies on the enzymatic synthesis and metabolism of folatecoenzymes in plants IV. (For the previous paper, Part III, seeReference (21)) . Part of this paper was presented at the AnnualMeeting of the Agricultural Chemical Society of Japan held atTokyo on April 4, 1967 (Received October 8, 1969; )     

Biosynthesis of alkane-2,3-diols: enzymatic reduction of 3-hydroxyoctadecane-2-one to octadecane-2,3-diol by a NADPH (B-side) specific microsomal reductase from the uropygial glands of ring-necked pheasants (Phasianus colchicus).

Cell-free preparations from the uropygial gland of ring-necked pheasant catalyzed the reduction of a synthetic R,S-mixture of 3-hydroxyl[3-¹⁴C]octadecane-2-one (acyloin) to a mixture of threo- and erythro-[3-¹⁴C]octadecane-2,3-diol, the final step in the postulated pathway for the biosynthesis of alkane-2,3-diols. The product of enzymatic reduction was identified by Chromatographic techniques and chemical degradation studies. The acyloin reductase showed a pH optimum near 4.0 and specificity for NADPH. With stereospecifically labeled [³H]NADPH, it was shown that acyloin reductase preferentially transferred hydride from the B-side of the nicotinamide ring to the acyloin. A typical Michaelis-Menten substrate saturation was observed for the acyloin and an apparent K_m of 70 μm was calculated from linear double reciprocal plots. Acyloin reductase was inhibited by thioldirected reagents such as p-chloromercuribenzoate and N-ethylmaleimide. Subcellular fractionation of the gland homogenates using sucrose density gradient centrifugation showed that acyloin reductase activity coincided with NADPH:cytochrome c reductase activity, strongly suggesting that acyloin reductase is localized in the microsomal membranes.     

Synthesis of 4-thia-[6-13C]lysine from [2-13C]glycine: access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine

4-Thialysine (S-(2-aminoethyl)-l-cysteine) is an analog of lysine. It has been used as an alternative substrate for lysine in enzymatic reactions. Site-directed isotopomers are often needed for elucidation of mechanism of reactions. 4-Thialysine can be synthesized by reacting cysteine with 2-bromoethylamine, an important reagent in chemical-modification rescue (CMR) of proteins. Here, we present the synthesis of 4-thia-[6-¹³C]lysine, one of the isotopomers of 4-thialysine, from commercially available starting material [2-¹³C]glycine via formation of five intermediates including 2-amino[2-¹³C]ethanol and 2-bromo[1-¹³C]ethylamine. The compounds were characterized using various spectroscopic techniques. Moreover, we discuss that our strategy would provide access to site-directed isotopomers of 2-aminoethanol, 2-bromoethylamine and 4-thialysine. Biological activity of 4-thia-[6-¹³C]lysine was tested in the enzymatic reaction of lysine 5,6-aminomutase.