Caveolin-rich lipid rafts of the plasma membrane of mature cerebellar granule neurons are microcompartments for calcium/reactive oxygen and nitrogen species cross-talk signaling

In previous works, we have shown that L-type voltage-operated calcium channels, N-methyl-d-aspartate receptors (NMDAr), neuronal nitric oxide synthase (nNOS) and cytochrome b₅ reductase (Cb₅R) co-localize within the same lipid rafts-associated nanodomains in mature cerebellar granule neurons (CGN). In this work, we show that the calcium transport systems of the plasma membrane extruding calcium from the cytosol, plasma membrane calcium pumps (PMCA) and sodium–calcium exchangers (NCX), are also associated with these nanodomains. All these proteins were found to co-immunoprecipitate with caveolin-1 after treatment with 25 mM methyl-β-cyclodextrin, a lipid rafts solubilizing agent. However, the treatment of CGN with methyl-β-cyclodextrin largely attenuated the rise of cytosolic calcium induced by l-glutamate through NMDAr. Fluorescence energy transfer imaging revealed that all of them are present in sub-microdomains of a size smaller than 200 nm, with a peripheral distribution of the calcium extrusion systems PMCA and NCX. Fluorescence microscopy images analysis revealed high calcium dynamic sub-microcompartments near the plasma membrane in fura-2-loaded CGN at short times after addition of l-glutamate. In addition, the close proximity between sources of nitric oxide (nNOS) and superoxide anion (Cb₅R) suggests that these nanodomains are involved in the fast and efficient cross-talk between calcium and redox signaling in neurons.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

L-type calcium channels and cytochrome b5 reductase are components of protein complexes tightly associated with lipid rafts microdomains of the neuronal plasma membrane

The presence of cytosolic calcium microcompartments in neurons is well established. L-type voltage calcium channels play a leading role in the rise of cytosolic calcium in the neuronal soma and are sensitive to redox modulation. In a recent work [Samhan-Arias, A.K., García-Bereguiaín, M.A., Martín-Romero, F.J. and Gutiérrez-Merino, C. (2009) Mol. and Cell. Neurosci. 40, 14–26], we have shown that cytochrome b₅ reductase, whose deregulation leads to an overshot of superoxide anion production at the neuronal plasma membrane that triggers apoptosis in primary cultures of cerebellar granule neurons in culture, forms a large mesh of redox centres associated with lipid rafts in these neurons. In this work, we have implemented the use of fluorescent antibodies as reagents for quantitative Förster resonance energy transfer measurements and analysis using fluorescence microscopy images of cerebellar granule neurons in culture. The results of this study show that L-type voltage-operated calcium channels are also enriched in lipid rafts associated protein microdomains at a distance between 10 and 100 nm from cytochrome b₅ reductase. The methodological improvements done in this work can be also valuable for the study of proteins compartmentalization within other subcellular microdomains in any cell type in culture.     

The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b(5) is relatively slow (k=6 x 10(2)M(-1)s(-1) at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2 x 10(7)M(-1)s(-1)) with an apparent overall equilibrium constant of 1 microM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.     

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

Cytochrome c is released from coupled mitochondria of yeast en route to acetic acid-induced programmed cell death and can work as an electron donor and a ROS scavenger

To gain insight into the processes by which acetic acid-induced programmed cell death (AA-PCD) takes place in yeast, we investigated both cytochrome c release from yeast mitochondria and mitochondrial coupling over the time course of AA-PCD. We show that the majority of cytochrome c release occurs early in AA-PCD from intact coupled mitochondria which undergo only gradual impairment. The released cytochrome c can be reduced both by ascorbate and by superoxide anion and in turn be oxidized via cytochrome c oxidase, thus working both as a ROS scavenger and a respiratory substrate. Late in AA-PCD, the released cytochrome c is degraded.     

Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners

In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b(5) from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b(5) from outer mitochondrial membrane allowed reaching expression level up to 10(4) nmol of the hemeprotein per liter of culture. Recombinant cytochrome b(5) from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b(5) from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b(5) was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b(5) an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b(5).     

Reduction of cytochrome b5 by NADPH-cytochrome P450 reductase

The reduction of mammalian cytochrome b5 (b5) by NADPH-cytochrome P450 (P450) reductase is involved in a number of biological reactions. The kinetics of the process have received limited consideration previously, and a combination of pre-steady-state (stopped-flow) and steady-state approaches was used to investigate the mechanism of b5 reduction. In the absence of detergent or lipid, a reductase-b5 complex is formed and rearranges slowly to an active form. Electron transfer to b5 is rapid within this complex (>30 s(-1) at 23 degrees C), as fast as to cytochrome c. With excess b5 present, a burst of reduction is observed, consistent with rapid electron transfer to one or two b5 molecules per reductase, followed by a subsequent rate-limiting event. In detergent vesicles, the reductase and b5 interact rapidly but electron transfer is slower (approximately 3 s(-1) at 23 degrees C). Experiments with dimyristyl lecithin vesicles yielded results intermediate between the non-vesicle and detergent systems. These steady-state and pre-steady-state kinetics provide views of the different natures of the reduction of b5 by the reductase in the absence and presence of vesicles. Without vesicles, the encounter of the reductase and b5 is rapid, followed by a slow reorganization of the initial complex (approximately 0.07 s(-1)), very fast reduction, and dissociation. In vesicles, encounter is rapid and the slow step (approximately 3 s(-1)) is reduction within a complex less favorable for reduction than in the non-vesicle systems.     

Effects of charged amino-acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045+/-0.009 nm and 0.088+/-0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b(5) surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b(5) complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b(5), do occur in the association between cytochrome b(5) and cytochrome c.     

Cytochrome b(5) is a major reductant in vivo of human indoleamine 2,3-dioxygenase expressed in yeast

The evolutionary relationship of indoleamine 2,3-dioxygenase (IDO) to some gastropod myoglobins suggests that IDO may undergo autoxidation in vivo such that one or more currently unidentified electron donors are required to maintain IDO heme iron in the active, ferrous state. To evaluate this hypothesis we have used yeast knockout mutants in combination with a recently developed yeast growth assay for IDO activity in vivo to demonstrate a role for cytochrome b(5) and cytochrome b(5) reductase in maintaining IDO activity in vivo.     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

Developmental shift in the apostat: comparison of neurones and astrocytes

The intrinsic pathway of apoptosis was investigated in cell-free extracts of neurones and astrocytes at various stages of maturation. Neuronal extracts were activated 65-fold after 3 days, 9-fold after 7 days, and were not activated after 10 days in culture. In contrast, astrocyte extracts were activated to a similar extent at all stages, up to 60 days in culture. The co-incubation of neuronal and astrocyte extracts followed by addition of cytochrome c/2'-deoxyadenosine 5'-triphosphate led to a 40-fold activation, suggesting that the development-associated neuronal shift does not involve the appearance of a dominant inhibitor, but rather downregulation of some key component(s) involved in caspase activation.     

The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

Mammalian mitochondrial and microsomal cytochromes b(5) exhibit divergent structural and biophysical characteristics

The only outer mitochondrial membrane cytochrome b(5) examined to date, from rat (rOM b(5)), exhibits greater stability than known mammalian microsomal (Mc) isoforms, as well as a much higher kinetic barrier for hemin dissociation and a more negative reduction potential. A BlastP search of available databases using the protein sequence of rOM b(5) as template revealed entries for analogous proteins from human (hOM b(5)) and mouse (mOM b(5)). We prepared a synthetic gene coding for the heme-binding domain of hOM b(5), and expressed the protein to high levels. The hOM protein exhibits stability, hemin-binding, and redox properties similar to those of rOM b(5), suggesting that they are characteristic of the OM b(5) subfamily. The divergence in properties between the OM and Mc b(5) isoforms in mammals can be attributed, at least in part, to the presence of two extended hydrophobic patches in the former. The biophysical properties characteristic of the OM proteins may be important in facilitating the two functions proposed for them so far, reduction of ascorbate radical and stimulation of androgen synthesis.     

Antioxidant Ascorbate Is Stabilized by NADH-Coenzyme Q10 Reductase in the Plasma Membrane

Plasma membranes isolated from K562 cells contain an NADH-ascorbate free radical reductase activity and intact cells show the capacity to reduce the rate of chemical oxidation of ascorbate leading to its stabilization at the extracellular space. Both activities are stimulated by CoQ₁₀ and inhibited by capsaicin and dicumarol. A 34-kDa protein (p34) isolated from pig liver plasma membrane, displaying NADH-CoQ₁₀ reductase activity and its internal sequence being identical to cytochrome b ₅ reductase, increases the NADH-ascorbate free radical reductase activity of K562 cells plasma membranes. Also, the incorporation of this protein into K562 cells by p34-reconstituted liposomes also increased the stabilization of ascorbate by these cells. TPA-induced differentiation of K562 cells increases ascorbate stabilization by whole cells and both NADH-ascorbate free radical reductase and CoQ₁₀ content in isolated plasma membranes. We show here the role of CoQ₁₀ and its NADH-dependent reductase in both plasma membrane NADH-ascorbate free radical reductase and ascorbate stabilization by K562 cells. These data support the idea that besides intracellular cytochrome b ₅-dependent ascorbate regeneration, the extracellular stabilization of ascorbate is mediated by CoQ₁₀ and its NADH-dependent reductase.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Reduced de novo synthesis of 5-methyltetrahydrofolate and reduced taurine levels in ethanol-treated chick brains

In previous studies, exogenous ethanol (3 mmol EtOH/kg egg) caused a 1.6-fold increase in chick brain homocysteine (HoCys) levels at 11 days of development and the mixture of 3 mmol EtOH/kg egg and 34 μmol folic acid/kg egg attenuated EtOH-induced increases in chick brain HoCys levels. Because HoCys is converted to methionine utilizing the methyl donor, 5-methyltetrahydrofolate (5-methyl THF), we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 μmol 5-methyl THF/kg egg inhibited chick brain 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH; EC 1.5.1.6) activities and brain N⁵, N¹⁰-methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) activities at 11 days of development. Three daily dosages of 3 mmol EtOH/kg egg (E_0–2) caused approximately a 7-fold reduction in brain 10-FTHF DH activities and approximately a 1.9-fold reduction in brain MTHFR activities as compared to controls at 11 days of development (p ≤ 0.05). Because HoCys is also removed by the transsulfuration pathway, which synthesizes taurine, we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 μmol 5-methyl THF/kg egg influenced chick brain taurine levels. In EtOH-treated and EtOH and 5-methyl THF-treated embryos, brain taurine levels decreased by approximately 5.5-fold and 6.2-fold as compared to controls, respectively (p ≤ 0.05). Exogenous 5-methyl THF failed to attenuate EtOH-induced decreased brain taurine levels at 11 days of development.