Effect of Folacin Deficiency on Folacin-binding Proteins in the Rat

Zamierowski, Marilyn M.; Wagner, Conrad

doi:10.1093/jn/107.10.1937

Cited by 33 publications

(10 citation statements)

References 21 publications

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“…Its high abundance in the liver (4) results in the major portion (if not all) of 5-methyltetrahydrofolate tightly bound to GNMT (8). It should be noted that GNMT is abundant in other tissues, e.g.…”

Section: Discussionmentioning

confidence: 99%

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

Luka

Pakhomova

Loukachevitch

et al. 2007

Journal of Biological Chemistry

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Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr 5 , Leu 207 , and Met 215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.

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Section: Discussionmentioning

confidence: 99%

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

Luka

Pakhomova

Loukachevitch

et al. 2007

Journal of Biological Chemistry

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“…Previously, lO-formyl-THF DH was identified as one of three polypeptides that can be isolated from rat liver cytosol on the basis of folate binding ability (3)(4)(5). This observed association with folate is directly related to the enzyme's catalysis of the oxidation of 10-formyl-THF to THF and CO2 in a NADP+-dependent reaction.…”

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confidence: 99%

Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice.

Champion

Cook

Tollaksen

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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During the analysis of liver protein expression in the offspring of male mice Irradiated with fissionspectrum neutrons, one ofprin displayed a heritable 50% decrease in the abundance of two proteins. Homozygous mice lacing detectable quantities of these proteins were obtained through breeding. Characterization of this protein deficiency has identified these liver proteins as forms of the enzyme 10-formyltetrahydrofolate dehydrogenase (10- Although the physical properties and substrate and product binding characteristics of 10-formyl-THF DH have been described, the physiological role of this enzyme in mammalian metabolism has not been directly addressed. Certain characteristics of mice lacking 10-formyl-THF DH indicate that disruption of the structural gene has significant physiological consequences. While homozygous mice appear completely normal despite this enzyme deficiency, breeding pairs of homozygotes show a dramatically decreased reproductive efficiency (6). Analysis of pool sizes of hepatic folate derivatives revealed shifts in the steady-state abundance of some forms of THF in mice lacking 10-formyl-THF DH. EXPERVIMENTAL PROCEDURESLiver Protein Sample Preparation. Liver protein samples were prepared as described (7) and were either frozen at -700C and later ground in liquid nitrogen or homogenized in 8 volumes of a solution containing 9 M urea, 2% 2-mercaptoethanol, 2% ampholytes (Pharmacia LKB, pH 9-11), and 4% Nonidet P-40. The homogenates were centrifuged at 435,000 x g for 5 min in a Beckman TL100 ultracentrifuge. The resulting supernatants containing denatured liver proteins were collected and stored frozen at -700C until analysis by two-dimensional electrophoresis (2DE).Preparation of Clarified Mouse Liver Cytosol. Mouse liver was minced with scissors and homogenized in 5 volumes (4-5 ml) of 0.25 M sucrose buffered with 10 mM Hepes using a motor-driven pestle at 3000 rpm for 30 strokes on ice. Aliquots (1 ml) of homogenate were then centrifuged for 15 min at room temperature in a Beckman TL100 ultracentrifuge. The supernatants were collected, combined, and dialyzed at 40C against 0.02 M phosphate buffer (pH 7.1), which was changed three times at hourly intervals.2DE. Aliquots (10 pIl) of mouse liver samples were separated in the first dimension by isoelectric focusing in 10-inch rod gels (8). A mixture of 25% Biolyte (Bio-Rad) pH 3-10/ 25% Servalyte pH 3-10/50%6 Biolyte pH 5-7 was used to generate the pH gradient. The second-dimension separation was performed as described by O'Farrell (9) on 10-17% linear gradient slab gels prepared as described (10). Gels were fixed

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“…Of these, the binder in the membrane of Lactobacillus casei appears to have such function (27). The rat intestinal epithelium (28) and liver plasma membranes (29) were reported to bind folic acid and its derivatives. In both tissues the activity found was much lower than that in the kidney, making it difficult to evaluate their function experimentally.…”

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confidence: 99%

Demonstration of high-affinity folate binding activity associated with the brush border membranes of rat kidney.

Selhub¹,

Rosenberg²

1978

Proc. Natl. Acad. Sci. U.S.A.

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Folate binding activity of high affinity was identified in the particulate fractions of rat kidney homogenates. This binding activity cofractionated with alkaline phosphatase and maltase, two brush border membranes markers. With an enriched preparation of brush border membranes, freed of endogenous folate by acid treatment, the binding of [3Hlfolate was found to be saturable (Kb = 4.2 X 10-11 M) and rapid. Binding was optimal at pH 6. 4-7.7 [3H]folic acid into the left renal artery of dogs, 6-24% of the 3H was still retained in the kidney and could be flushed out into the urine by a large dose of unlabeled folic acid. Rubin et al. (3) showed that methotrexate, which inhibits folic acid reabsorption (1, 2), is taken up by rabbit renal cortical slices by two processes: one which is rapid and energy independent and a second which is slower and requires energy.On the basis of these observations it has been suggested (3, 4) that this initial step of folate reabsorption in the kidney involves the binding of filtered folate to a specific protein of the proximal tubular cells. Dihydrofolate reductase has been proposed as the binding protein because this enzyme is present in relative abundance in this tissue. An alternate possibility is that this cellular factor is a high-affinity binding protein of the type that has been recently identified in milk (5), blood serum (6-9), and various other tissues including the soluble fraction of porcine kidney homogenates (10-13). Such proteins are characterized by rapid association and slow dissociation with folic acid (Kb = 10-1o-10-11 M), a preference for folate mono-and polyglutamates over those with one carbon substitution at the 5 position or methotrexate (7), and different effects of pH on binding of folic acid and N5-methyltetrahydrofolate (14, 15 Schmitz et al. (17). In this procedure, CaCl2 was added to the homogenate to a final concentration of 10 mM. The mixture was stirred for 10 min and centrifuged at 2000 X g for 10 min, producing a "cell debris" precipitate (fraction P1). Centrifugation of the supernatant at 20,000 X g for 15 min resulted in the separation of soluble proteins (fraction S) from a second pellet (fraction P2). By electron microscopy, P2 appeared to be a highly enriched preparation of membrane vesicles. All fractions were assayed for protein (18), their capacity to bind radioactive folate (see below), and markers activities for brush border membranes (alkaline phosphatase and maltase) (19,20), mitochondria (succinate cytochrome c reductase) (21), basolateral membranes (Na+,K+-ATPase) (22), microsomes (glucose-6-phosphatase), and lysosomes (acid phosphatase) (23)

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Effect of Folacin Deficiency on Folacin-binding Proteins in the Rat

Cited by 33 publications

References 21 publications

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice.

Demonstration of high-affinity folate binding activity associated with the brush border membranes of rat kidney.

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