Stimulation of brain aromatic alkylamine N-methyltransferase activity by FAD and methylcobalamin

We studied the effects of methylcobalamin alone and with various reducing systems (FADH₂, FAD or its analogues in combination with NADH or β-mercaptoethanol) on the activity of partially purified aromatic alkylamine N-methyltransferase (AANMT) from rat brain. As expected, the specific activity of AANMT in the presence of methylcobalamin alone was enhanced (125% of control). Surprisingly, in the presence of FAD alone (but not FADH₂$\text{et cetera}$) it was 175% of control; and int the presence of methylcobalamin plus FAD plus β-mercaptoethanol it reached 307% of control. Preliminary evidence suggests that FAD induces allosteric kinetics for the activated enzyme with respect to the methyl donor 5-methyltetrahydrofolic acid (5-MTHF).     

Thermolabile 5,10-methylenetetrahydrofolate reductase as a cause of mild hyperhomocysteinemia.

Thermolability of 5,10-methylenetetrahydrofolate reductase (MTHFR) was examined as a possible cause of mild hyperhomocysteinemia in patients with premature vascular disease. Control subjects and vascular patients with mild hyperhomocysteinemia and with normohomocysteinemia were studied. The mean (+/- SD) specific MTHFR activity in lymphocytes of 22 control subjects was 15.6 (+/- 4.7) nmol CH2O/mg protein/h (range: 9.1-26.6), and the residual activity (+/- SD) after heat inactivation for 5 min at 46 degrees C was 55.3 (+/- 12.0)% (range: 35.9-78.3). By measurement of MTHFR activity, two distinct subgroups of hyperhomocysteinemic patients became evident. One group (n = 11) had thermolabile MTHFR with a mean (+/- SD) specific activity of 8.7 (+/- 2.1) nmol CH2O/mg protein/h (range: 5.5-12.7) and a residual activity, after heat inactivation, ranging from 0% to 33%. The other group (n = 28) had normal specific activity (+/- SD) of 21.5 (+/- 7.2) nmol CH2O/mg protein/h (range: 10.0-39.0) and a normal residual activity (+/- SD) of 53.8 (+/- 9.2)% (range: 33.1-71.5) after heat inactivation. The mean (+/- SD) specific activity of 29 normohomocysteinemic patients was 20.7 (+/- 6.5) nmol CH2O/mg protein/h (range: 9.4-33.8), and the mean (+/- SD) residual activity after heat inactivation was 58.2 (+/- 10.2)% (range: 43.0-82.0). Thus, in 28% of the hyperhomocysteinemic patients with premature vascular disease, abnormal homocysteine metabolism could be attributed to thermolabile MTHFR.     

Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase.

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.     

Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum

Methylenetetrahydrofolate reductase in Clostridium formicoaceticum has been purified to a specific activity of 140 mumol min-1 mg-1 when assayed at 37 degrees C, pH 7.2, in the direction of oxidation of 5-methyltetrahydrofolate with benzyl viologen as electron acceptor. The purified enzyme is judged to be homogeneous by polyacrylamide disc-gel electrophoresis and gel filtration. The enzyme which is an octamer has a molecular weight of about 237,000 and consists of four each of two different subunits having the molecular weights 26,000 and 35,000. The octameric enzyme contains per mol 15.2 +/- 0.3 iron, 2.3 +/- 0.2 zinc, 19.5 +/- 1.3 acid-labile sulfur, and 1.7 FAD. The UV-visible absorbance spectrum has a peak at 385 nm and a shoulder at 430 nm and is that of a flavoprotein containing iron-sulfur centers. The reductase, which is sensitive to oxygen, must be handled anaerobically and is stabilized by 2 mM dithionite. It catalyzes the reduction of methylene blue, menadione, benzyl viologen, rubredoxin, and FAD with 5-methyltetrahydrofolate and the oxidation of reduced ferredoxin and FADH2 with 5,10-methylenetetrahydrofolate. No activity was observed with pyridine nucleotides. It is suggested that the physiologically important reaction catalyzed by the enzyme is the reduced ferredoxin-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.     

Purification and properties of a NADH-dependent 5,10-methylenetetrahydrofolate reductase from Peptostreptococcus productus

The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5). The apparent Km for NADH was near 10 microM. The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far.     

High-performance liquid chromatographic measurement of 5,10-methylenetetrahydrofolate in liver.

The folate coenzyme 5,10-methylenetetrahydrofolate is an important folate metabolite which cannot be determined directly by HPLC near neutral pH because it dissociates to formaldehyde and tetrahydrofolate. A method for the determination of 5,10-methylenetetrahydrofolate in liver is described. This method involves (1) determination of liver 5-methyltetrahydrofolate; (2) chemical reduction of liver 5,10-methylenetetrahydrofolate (stabilized at pH 10) to 5-methyltetrahydrofolate; and (3) determination of total liver 5-methyltetrahydrofolate. Subtraction of (1) from (3) gives the concentration of 5,10-methylenetetrahydrofolate in liver.     

Polymorphisms of the 5,10-methylenetetrahydrofolate and the methionine synthase reductase genes as independent risk factors for spina bifida

We analyzed the role of the C677T polymorphism of the 5,10-methylenetetrahydrofolate and the A66G polymorphism of the methionine synthase reductase genes as risk factors for occurrence of spina bifida. The studied population included 106 mothers and 104 children from affected families, and a control group of 100 adults. We found statistically significant differences between the occurrence of the homozygosity in these polymorphisms in the groups of mothers and children with thoracolumbal defects (C677T polymorphism) and lumbosacral defects (A66G polymorphism). We postulate that these polymorphisms should be regarded as independent risk factors for spina bifida.     

The effect of Raney nickel on the covalent thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate complex.

Raney nickel (Ni(H)) catalyzes a specific reductive cleavage of carbon-sulfur bonds and, therefore, can be used to determine whether compounds are covalently bound to proteins through a sulfide linkage. When the covalent thymidylate synthetase-[3H]5-fluoro-2'-deoxyuridylic acid-[14C]-5,10-CH2H4-folate complex (Langenbach et al. (1972a), Biochem, Biophys. Res. Commun. 48, 1565) was denatured and then shaken with Ni(H) at 25 degrees C, both isotopes were rapidly cleaved from the protein, with identical reaction halftimes of less than 10 min. The liberated radioactivity was filterable through nitro-cellulose filters and comigrated with small molecules on Sephadex G-25. Both labels migrated identically upon paper chromatography. A [3H]5-fluoro-2'-deoxyuridylic acid-[35S]thymidylate synthetase complex was formed with enzyme isolated from Lactobacillus casei grown in the presence of [35S]cysteine. This complex, upon Ni(H) treatment, released both tritium and sulfur-35 at identical rates. Control experiments on amino acids showed that only the sulfur-containing amino acids are degraded by Ni(H). Cysteine was rapidly converted to alanine and methionine to alpha-aminobutyric acid. 5-Carboxymethylcysteine and 5-uracilylcysteine, simple models for the tenary enzyme-5-fluoro-2'-deoxyuridylic acid-5,10-CH2H4-folate complex, were converted to alanine at the same rate that 5-fluoro-2'-deoxyuridylic acid (FdUrd-5'-P) was cleaved from the enzyme. Native ribonuclease, which has a tightly coiled structure, was not affected by the reagent, but carboxymethylated ribonuclease was desulfurized. Amino acid analysis of Ni(H)-treated thymidylate synthetase showed that cysteine was the only amino acid degraded. Gel electrophoresis of the proteins after exposure to Ni(H) showed no breakage of polypeptide chains. These results support a sulfide linkage between FdUrd-5'-P and thymidylate synthetase in the covalent complex.     

Interaction of Cibacron Blue F3G-A and Procion Red HE-3B with sheep liver 5,10-methylenetetrahydrofolate reductase

Cibacron Blue F3G-A, a probe used to monitor nucleotide binding domains in enzymes, inhibited sheep liver 5,10-methylenetetrahydrofolate reductase competitively with respect to 5-methyltetrahydrofolate and NADPH. TheK _i values obtained by kinetic methods and theK _d value for the binding of the dye to the enzyme estimated by protein fluorescence quenching were in the range 0.9–1.2 μM. Another triazine dye, Procion Red HE-3B interacted with the enzyme in an essentially similar manner to that observed with Cibacron Blue F3G-A. These results as well as the interaction of the dye with the enzyme monitored by difference spectroscopy and intrinsic protein fluorescence quenching methods indicated that the dye was probably interacting at the active site of the enzyme by binding at a hydrophobic region.     

Purification and characterization of NADP(+)-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Peptostreptococcus productus marburg.

The 5,10-methylenetetrahydrofolate dehydrogenase of heterotrophically grown Peptostreptococcus productus Marburg was purified to apparent homogeneity. The purified enzyme catalyzed the reversible oxidation of methylenetetrahydrofolate with NADP+ as the electron acceptor at a specific activity of 627 U/mg of protein. The Km values for methylenetetrahydrofolate and for NADP+ were 27 and 113 microM, respectively. The enzyme, which lacked 5,10-methenyltetrahydrofolate cyclohydrolase activity, was insensitive to oxygen and was thermolabile at temperatures above 40 degrees C. The apparent molecular mass of the enzyme was estimated by gel filtration to be 66 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a single subunit of 34 kDa, accounting for a dimeric alpha 2 structure of the enzyme. Kinetic studies on the initial reaction velocities with different concentrations of both substrates in the absence and presence of NADPH as the reaction product were interpreted to indicate that the enzyme followed a sequential reaction mechanism. After gentle ultracentrifugation of crude extracts, the enzyme was recovered to greater than 95% in the soluble (supernatant) fraction. Sodium (10 microM to 10 mM) had no effect on enzymatic activity. The data were taken to indicate that the enzyme was similar to the methylenetetrahydrofolate dehydrogenases of other homoacetogenic bacteria and that the enzyme is not involved in energy conservation of P. productus.     

5-Methyltetrahydrofolate is photosensitive in the presence of riboflavin.

5-Methyltetrahydrofolate (5MTHF) is the main form of folate in human plasma, and an important vitamin for human health. Photodegradation of folates may have played a role in the development of different human skin colours. 5MTHF can be degraded directly by exposure to ultraviolet radiation or by exposure to visible light in the presence of endogenous sensitizers like riboflavin (RF). These photochemical reactions were studied by absorption spectroscopy. While 5MTHF is stable under UV and visible light exposure in pure aqueous media, it is quickly degraded in the presence of RF during UVA and blue light exposure. The degradation of 5MTHF is dependent on the concentration of RF, but not on the concentration of 5MTHF itself. UVA and blue light gave similar reactions. Further investigations are necessary to evaluate the consequences of large light exposures in vivo in humans. Our findings should be taken into the ongoing discussion about the development of human skin colours. Due to the presence of RF in human blood, folate can be significantly degraded during prolonged or intense blue light exposure. Thus, a dark skin colour may be favourable for prevention of folate degradation under high solar fluence rates, such as in equatorial areas.     

Activity of an NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase in normal tissue, neoplastic cells, and oncogene-transformed cells

As an extension of the previously reported observation concerning the existence of NAD-dependent 5,10-methylenetrahydrofolate dehydrogenase in transformed cells a variety of tissues and cell lines have been assayed for this activity. This activity was found in all assayed transformed cells. Results with rat liver derived epithelial (RLE) cells transformed with a series of oncogenes (v-raf, v-raf/v-myc (J2), v-myc (J5), and v-Ha-ras (pRNR16)) indicated that expression of activity correlates with the extent of transformation and was independent of the oncogene used for transformation. Compared to previously reported values for normal tissue, surprisingly high levels of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase were found in the rat adrenal cortex. This activity was not seen in mouse or bovine adrenal. Enzymatic activity was also detected in mouse bone marrow and was strain dependent. The levels of activity in mouse bone marrow were lower than previously reported. The NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase activity in rat adrenal and RLE cells may represent tools for studying the regulation of expression of this activity.     

Ferrisiderophore reductase activity associated with an aromatic biosynthetic enzyme complex in Bacillus subtilis

The cytoplasmic fractions obtained from Bacillus subtilis strains W168 and WB2802 catalyzed reductive release of iron from the ferric chelate of 2,3-dihydroxybenzoic acid (ferri-DHB), the ferrisiderophore produced by B. subtilis. Ferrisiderophore reductase activity may insert iron into metabolism. This activity required a reductant (reduced nicotinamide adenine dinucleotide phosphate was preferred), was oxygen sensitive, and was stimulated by flavin mononucleotide plus certain divalent cations. The cytoplasmic fractions also reduced 2,6-dichlorophenolindophenol; this reaction was stimulated by flavin mononucleotide plus a divalent cation. Ferri-DHB and 2,6-dichlorophenolindophenol reductase activities were copurified by phosphocellulose and diethylaminoethyl-cellulose chromatography. Nondenaturing polyacrylamide gel electrophoresis of the purified material revealed that both ferri-DHB and 2,6-dichlorophenolindophenol reductase activities were located in a protein band at Rf 0.75. The chromatographic procedures purified a reductase known to be associated with two aromatic biosynthetic enzymes, chorismate synthase and dehydroquinate synthase. Therefore, a portion of the ferrisiderophore reductase activity in B. subtilis may be catalyzed by a reductase that also is essential for aromatic biosynthesis.     

Cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity.

The enzyme 5-formyl tetrahydrofolate cyclodehydrase plays an important role in the conversion of 5-formyl tetrahydrofolate to 5,10-methenyl tetrahydrofolate. A second enzyme, cyclohydrolase, converts 5,10-methenyl tetrahydrofolate to 10-formyl tetrahydrofolate. These folate derivatives play a significant part in the biosynthesis of purines. A method has been devised for the cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity which uses 5-formyl tetrahydrofolate or 5,10-methenyl tetrahydrofolate as substrate respectively, blocking possible interferences by other enzymes, and allows the nonenzymatic reduction of nitro-blue tetrazolium by 5,10-methenyl tetrahydrofolate formed by the action of the cyclodehydrase on the substrate 5-formyl tetrahydrofolate, and by 10-formyl tetrahydrofolate formed by the action of cyclohydrolase on the substrate 5,10-methenyl tetrahydrofolate, thus revealing intracellular sites of enzyme activity. The methods appear to show only intracellular localization of the blue formazan deposits of reduced tetrazolium. The distribution of positivity in cells of human blood and bone marrow is described.     

Purification, characterization, cloning, and amino acid sequence of the bifunctional enzyme 5,10-methylenetetrahydrofolate dehydrogenase/5,10-methenyltetrahydrofolate cyclohydrolase from Escherichia coli.

We have purified the enzyme 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) from Escherichia coli to homogeneity by a newly devised procedure. The enzyme has been purified at least 2,000-fold in a 31% yield. The specific activity of the enzyme obtained is 7.4 times greater than any previous preparation from this source. The purified enzyme is specific for NADP. The protein also contains 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9) activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behavior on a molecular sieving column suggest that the enzyme is a dimer of identical subunits. We have cloned the E. coli gene coding for the enzyme through the use of polymerase chain reaction based on primers designed from the NH2 terminal analysis of the isolated enzyme. We sequenced the gene. The derived amino acid sequence of the enzyme contains 287 amino acids of Mr 31,000. The sequence shows 50% identity to two bifunctional mitochondrial enzymes specific for NAD, and 40-45% identity to the presumed dehydrogenase/cyclohydrolase domains of the trifunctional C1-tetrahydrofolate synthase of yeast mitochondria and cytoplasm and human and rat cytoplasm. An identical sequence of 14 amino acids with no gaps is present in all 7 sequences.     

Effect of direct suppression of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site on the interconversion of tetrahydrofolate cofactors to dihydrofolate by antifolates. Influence of degree of dihydrofolate reductase inhibition.

An important unresolved issue in antifolate pharmacology is the basis for the observation that the major portion of cellular tetrahydrofolate cofactors is preserved after dihydrofolate reductase activity is abolished by antifolates despite the fact that tetrahydrofolate cofactor-dependent purine and pyrimidine biosynthesis ceases. This has been attributed to feedback inhibition of thymidylate synthase by dihydrofolate polyglutamates that accumulate in the presence of antifolates. This report combines network thermodynamic modeling and experimental observations to evaluate the effects of direct inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site with a potent lipophilic quinazoline antifolate PD130883 on folate oxidation in cells. Computer simulations predict and the data indicate that marked PD130883 suppression of thymidylate synthase only slows the rate but not the extent of tetrahydrofolate cofactor interconversion to dihydrofolate upon complete suppression of dihydrofolate reductase with trimetrexate. These observations are consistent with earlier studies from this laboratory with fluorodeoxyuridine inhibition at the deoxyuridylate binding site. Hence, the much weaker inhibition by dihydrofolate polyglutamates at the level of thymidylate synthase cannot account for the apparent preservation of tetrahydrofolate cofactor pools in cells and has virtually no pharmacologic significance under conditions in which antifolates completely suppress dihydrofolate reductase. The extent of interconversion of tetrahydrofolate cofactors to dihydrofolate is strongly influenced by residual dihydrofolate reductase catalytic activity. Exposure of cells to 0.1 microM trimetrexate results in only approximately 60% of maximum dihydrofolate levels achieved when dihydrofolate reductase activity is abolished. Network thermodynamic simulations predict, and experiments verify, that inhibition of thymidylate synthase at the 5,10-methylenetetrahydrofolate site by PD130883, when dihydrofolate reductase is only partially suppressed (approximately 85%) with 0.1 microM trimetrexate, substantially decreases (31-47%) the net level of interconversion of tetrahydrofolate cofactors to dihydrofolate. Further computer simulations predict that under conditions in which residual dihydrofolate reductase activity persists within the cells (more than about 5%), feedback inhibitory effects of dihydrofolate polyglutamates as well as other weak inhibitors of thymidylate synthase can significantly limit the extent of net interconversion of tetrahydrofolate cofactors to dihydrofolate and produce an apparent "compartmentation phenomenon" in which tetrahydrofolate cofactor pools are preserved within the cell in the presence of antifolates. Residual dihydrofolate reductase activity cannot, however, account for the partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure to high trimetrexate or methotrexate levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct assay method for guanosine 5'-monophosphate reductase activity.

A sensitive and simple micromethod for the accurate measurement of GMP reductase (EC 1.6.6.8) activity in crude extracts is described. The reaction product of [8-14C]IMP was separated from the substrate [8-14C]GMP by descending chromatography on Whatman DE81 ion-exchange paper. This separation method provides an analysis of the possible interfering reactions, such as the metabolic conversion of the substrate GMP to GDP, GTP, and/or guanosine, and guanine and the loss of the product IMP to inosine, hypoxanthine, and other metabolites. Low blank values (70-90 cpm) were obtained consistently with this assay because the IMP spot moves faster than the GMP spot. The major advantages of this method are direct measurement of GMP reductase activity in crude extracts, high sensitivity (with a limit of detection of < 10 pmol of IMP production), high reproducibility (< +/- 5%), and capability to measure activity in small samples (9 micrograms protein).     

Radioenzymatic assay for reductive catalysis of N(5)N(10)-methylenetetrahydrofolate by methylenetetrahydrofolate reductase

Methylenetetrahydrofolate reductase catalyzes the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate. Because this substrate is unstable and dissociates spontaneously into formaldehyde and tetrahydrofolate, the customary method to assay the catalytic activity of this enzyme has been to measure the oxidation of [14C]N(5)-methyltetrahydrofolate to N(5), N(10)-methylenetetrahydrofolate and quantify the [14C]formaldehyde that dissociates from this product. This report describes a very sensitive radioenzymatic assay that measures directly the reductive catalysis of N(5),N(10)-methylenetetrahydrofolate. The radio-labeled substrate, [14C]N(5),N(10)-methylenetetrahydrofolate, is prepared by condensation of [C(14)]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained for several months by storage at -80 degrees C in a pH 9.5 buffer. Partially purified methylenetetrahydrofolate reductase from rat liver, incubated with the radio-labeled substrate and the cofactors, NADPH and FAD at pH 7. 5, generates [14C]N(5)-methyltetrahydrofolate, which is stable and partitions into the aqueous phase after the assay is terminated with dimedone and toluene. A K(m) value of 8.2 microM was obtained under conditions of increasing substrate concentration to ensure saturation kinetics. This method is simple, very sensitive and measures directly the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate, which is the physiologic catalytic pathway for methylenetetrahydrofolate reductase.