Robert J. Cook

Effect of nitrous oxide inactivation of vitamin B12-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver☆

The effects of nitrous oxide inactivation of the vitamin B12-dependent enzyme, methionine synthetase (EC 2.1.1.13), on the subcellular distribution of hepatic folate coenzymes was determined. In controls, cytosolic folates were 5-methyltetrahydrofolate (45%), 5- and 10-formyltetrahydrofolate (9 and 19%, respectively), and tetrahydrofolate (27%). Exposure of rats to an atmosphere containing 80% nitrous oxide for 18 h resulted in a marked shift in this distribution pattern to 5-methyltetrahydrofolate, 84%; 5- and 10-formyltetrahydrofolate, 2.1 and 9.1%, respectively; and tetrahydrofolate, 4.7%. Activity of the cytosolic enzyme, methionine synthetase, was reduced by about 84% as compared to that of air breathing controls. In controls, mitochondrial folates were 5-methyltetrahydrofolate (7.3%), 5- and 10-formyltetrahydrofolate (11.5 and 33.1%, respectively), and tetrahydrofolate (48.1%). This distribution did not change after exposure to nitrous oxide. These results show that the effects of nitrous oxide inactivation of vitamin B12 are confined to the cytosol, at least in the short term, and suggest that there is little, if any, transport of free folates between the cytosolic and mitochondrial compartments.

Enzymatic properties of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are flavoproteins which catalyze the oxidative demethylation of dimethylglycine to sarcosine and sarcosine to glycine, respectively. During these reactions tightly bound tetrahydropteroylpentaglutamate (H4PteGlu5) is converted to 5,10-methylene tetrahydropteroylpentaglutamate (5,10-CH2-H4PteGlu5), although in the absence of H4PteGlu5, formaldehyde is produced. Single turnover studies using substrate levels of the enzyme (2.3 microM) showed pseudo-first-order kinetics, with apparent first-order rate constants of 0.084 and 0.14 s-1 at 23 and 48.3 microM dimethylglycine, respectively, for dimethylglycine dehydrogenase and 0.065 s-1 at 47.3 microM sarcosine for sarcosine dehydrogenase. The rates were identical in the absence or presence of bound tetrahydropteroylglutamate (H4PteGlu). Titration of the enzymes with substrate under anaerobic conditions did not disclose the presence of an intermediate semiquinone. The effect of dimethylglycine concentration upon the rate of the dimethylglycine dehydrogenase reaction under aerobic conditions showed nonsaturable kinetics suggesting a second low-affinity site for the substrate which increases the enzymatic rate. The Km for the high-affinity active site was 0.05 mM while direct binding for the low-affinity site could not be measured. Sarcosine and dimethylthetin are poor substrates for dimethylglycine dehydrogenase and methoxyacetic acid is a competitive inhibitor at low substrate concentrations. At high dimethylglycine concentrations, increasing the concentration of methoxyacetic acid produces an initial activation and then inhibition of dimethylglycine dehydrogenase activity. When these compounds were added in varying concentrations to the enzyme in the presence of dimethylglycine, their effects upon the rate of the reaction were consistent with the presence of a second low-affinity binding site on the enzyme which enhances the reaction rate. When sarcosine is used as the substrate for sarcosine dehydrogenase the kinetics are Michaelis-Menten with a Km of 0.5 mM for sarcosine. Also, methoxyacetic acid is a competitive inhibitor of sarcosine dehydrogenase with a Ki of 0.26 mM. In the absence of folate, substrate and product determinations indicated that 1 mol of formaldehyde and of sarcosine or glycine were produced for each mole of dimethylglycine or sarcosine consumed with the concomitant reduction of 1 mol of bound FAD.

Measurement of a folate binding protein from rat liver cytosol by radioimmunoassay

Abstract A radioimmunoassay has been developed for the folate binding protein from rat liver cytosol with a molecular weight of 150,000 which was recently purified to homogeneity (Suzuki, N., and Wagner, C., 1980, Arch. Biochem. Biophys. 199 , 236–248). This method has indicated that the binding protein (FBP-CII) is found primarily in the liver. A significant amount of FBP-CII was also found in the kidney and much reduced levels in spleen, serum, brain, lung, and heart. No FBP-CII could be detected in small intestine, skeletal muscle, or testes. Small amounts of cross-reacting material were found in the livers of mouse, dog, chick, and humans. Levels of FBP-CII were not decreased in the livers of folate-deficient rats. Assays of rat fetal liver and kidney 2 days prior to birth showed much lower levels which increased rapidly at birth. These data are consistent with the FBP-CII fulfilling a role as a folate storage protein in rat liver.

[41] Dimethylglycine dehydrogenase and sarcosine dehydrogenase: Mitochondrial folate-binding proteins from rat liver☆

Publisher Summary This chapter describes assay, purification, and properties of dimethylglycine dehydrogenase and sarcosine dehydrogenase. These enzymes perform sequential oxidative demethylation reactions in the choline degradation pathway, which occurs exclusively in liver mitochondria. Both enzymes contain covalently bound flavin adenine dinucleotide (FAD), and tightly, but not covalently, bound H 4 PteGlu 5 . The activity of these two enzymes may be estimated by linking the reduction of FAD to phenazine methosulfate (PMS) and then following the reduction of 2,6-dichlorophenolindophenol (DCPIP) at 600 nm. Enzyme assays of crude tissue extracts or mitochondrial sonicates require the addition of potassium cyanide (KCN) to inhibit nonspecific reduction of the dye. Substrate blanks should also be assayed under these conditions. The assay is run at room temperature. The purification procedure is for 200–300 g wet wt. of rat liver and is a modified version of Wittwer and Wagner. These two enzymes show typical flavoprotein UV-visible absorption spectra. The flavin is covalently linked via the 8α position of the isoalloxazine ring to an imidazole N(3) of a histidine residue. Both enzymes are single polypeptides, and comparison of the amino acid compositions indicates a high degree of structural homology.

[40] Purification of rat liver folate-binding protein: cytosol I

Publisher Summary This chapter describes assay, purification, and properties of folate-binding protein: cytosol I (FBP-CI) from rat liver. During the initial stages of purification FBP-CI is detected by virtue of the naturally bound ligand, [ 3 H]H 4 PteGlu 5 . Subsequent stages in the purification procedure release the bound ligand and the protein is then detected by its ability to bind the released H 4 PteGlu 5 . Binding of [ 3 H]folates is measured by a centrifugal procedure, which rapidly separates macromolecules from ligand using BioGel P-2 (Bio-Rad) columns. Protein may also be desalted prior to measuring binding of [ 3 H]folates by this method. FBP-CI has a native M R of 210,000 as determined by gel filtration. Pure protein gives a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) with a M r of 100,000, suggesting FBP-CI is a dimer. The endogenous folate ligand is tightly bound H 4 PteGlu 5 . Binding studies showed that the fully reduced folate monoglutamates are most effective at competing with the natural ligand, H 4 PteGlu 5 , for binding, while PteGlu is 20-fold less effective.

Covalent binding of folic acid to dimethylglycine dehydrogenase

Dimethylglycine dehydrogenase (EC 1.5.99.2) carries out the oxidative demethylation of dimethylglycine to sarcosine in liver mitochondria. In vivo, the enzyme uses tightly bound tetrahydropteroyl pentaglutamate (H4PteGlu5) as an acceptor of the one-carbon group generated during the reaction. The purified enzyme can use, but does not require, H4PteGlu5 and under these conditions formaldehyde is the one-carbon unit produced. It is reported that folic acid may be covalently linked to dimethylglycine dehydrogenase in a specific and saturable manner so that only 1 mole of folic acid is bound per mole of enzyme. Covalently bound folic acid blocks the subsequent binding of H4PteGlu, and does not inhibit the rate of dimethylglycine dehydrogenase activity in vitro.

IDENTIFICATION OF PROTEIN-ARGININE N-METHYLTRANSFERASE AS 10-FORMYLTETRAHYDROFOLATE DEHYDROGENASE

S-Adenosylmethionine:protein-arginineN-methyltransferase (EC 2.1.1.23; protein methylase I) transfers the methyl group ofS-adenosyl-l-methionine to an arginine residue of a protein substrate. The homogeneous liver protein methylase I was subjected to tryptic digestion followed by reverse phase high performance liquid chromatography (HPLC) separation and either “on-line” mass spectrometric fragmentation or “off-line” Edman sequencing of selected fractions. Data base searching of both the mass spectrometric and Edman sequencing data from several peptides identified the protein methylase as 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6; Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965–4973; Swiss accession number P28037). This identification was confirmed by comparative HPLC tryptic peptide mapping and affinity chromatography of the methylase on the 5-formyltetrahydrofolate-Sepharose affinity gel used to purify the dehydrogenase. The purified rat liver methylase had approximately 33% of the 10-formyltetrahydrofolate dehydrogenase and 36% of the aldehyde dehydrogenase activity as compared with the recombinant dehydrogenase, which also had protein methylase I activity. Polyclonal antibodies against recombinant dehydrogenase reacted with protein methylase I purified either by polyacrylamide gel electrophoresis or 5-formyltetrahydrofolate affinity chromatography. In each instance there was only a single immunoreactive band at a molecular weight of ∼106,000. Together, these results confirm the co-identity of protein-arginine methyltransferase and 10-formyltetrahydrofolate dehydrogenase.

Use of 10-formyl-5,8-dideazafolate as substrate for rat 10-formyltetrahydrofolate dehydrogenase.

Publisher Summary This chapter discusses the use of 10-formyl-5,8-dideazafolate as substrate for rat 10-formyltetrahydrofolate dehydrogenase. 10-formyltetrahydrofolate (10-HCO-H 4 PteGlu) is susceptible to oxidative degradation and must be protected by reducing agents during synthesis and use in enzyme assays. The (6R,S)-10-HCO-H4PteGlu, while highly unstable, is easily generated from stable, commercially available (6R,S)-5-HCO-HaPteGlu in the presence of 2-mercaptoethanol (2-ME), using the method of Rabinowitz. The 10-formyl-5,8-dideazafolate was originally synthesized as a quinazoline analog of folic acid. It was found to be a modest inhibitor of rat liver dihydrofolate reductase and had activity against L1210 leukemia in mice. The oxidation of 10-formyl-5,8-dideazafolate is followed by the production of 5,8-dideazafolate at 295 nm 6 or NADPH at 340 nm. The absorption of 5,8-dideazafolate at 340 nm is reflected in the adjusted extinction coefficient for NADPH. The absorption of NADPH at 295 nm is approximately 2% of the extinction coefficient for 5,8-dideazafolate and is ignored. Hydrolase activity is followed by the production of 5,8-dideazafolate at 295 nm in the presence of 100 m M 2-ME. The assay may be run at room temperature. Assays of crude extracts require the removal of low molecular weight compounds by spin column desalting.