Experiments on the biological activities of various fractions of the folic acid antagonist "X-methyl" folic acid

A crude synthetic preparation called crude "X-methyl" folate has previously been shown to function as a folate antagonist for rats and chicks. This product has been shown to contain two folate antagonists: 9-methyl folate, present as 6% by weight of the product and which has low activity as a folate antagonist for Streptococcus faecalis, and pyrrofolic acid, a compound present in small amounts (0.04%), but having high anti-folate biological activity for S. faecalis. These experiments deal with the antifolate activity of these two fractions for the rat as measured by their effects on histidine oxidation. Rats were fed a purified diet based on 20% vitamin-free casein and containing 1.0% sulfasuxidine. When this diet was supplemented with a marginal amount of folic acid (0.3 mg per kg diet), the addition of 4 g of crude antagonist decreased histidine oxidation and decreased liver folate levels. The addition of 240 mg of pure 9-methyl folic acid (amount of 9-methyl folic acid in 4 g of crude) produced similar decreases in histidine oxidation and liver folate levels. A concentrate of pyrrofolic acid (equivalent to 4 g of crude) free of 9-methyl folic acid produced no decrease in histidine oxidation and minimal changes in liver folate. This indicates that the folate antagonist activity observed previously with animals is probably due to the 9-methyl folic acid component rather than to the pyrrofolic acid activity.     

Evidence for a second chemotactic system in the cellular slime mold, Polysphondylium.

An unidentified substance(s) in a commercial guanosine 3′,5′-cyclic monophosphoric acid (cyclic 3′,5′-GMP) preparation is effective in attracting the aggregating amoebae of the cellular slime mold, Polysphondylium pallidum. Bacterial extracts (Escherichia coli) and amoeba extracts (P. pallidum) attract both vegetative and aggregating amoebae. A crude enzyme preparation from amoebae is capable of reducing the chemotactic activity of the extracts on aggregating amoebae and eliminating the activity of the unknown substance in the commercial cyclic 3′,5′-GMP preparation. As only the extracts were shown to contain folic acid, and since the enzyme does not reduce folic acid activity, it is suggested that the extracts contain a factor (possibly folic acid) primarily active on vegetative amoebae and an acrasin. The commercial cyclic 3′,5′-GMP preparation contains only an acrasin. The acrasin is heat stable and nondialyzable.     

Separation of Folic Acid Reductase from Streptococcus faecium (ATCC 8043)

A strain of Streptococcus faecium (ATCC 8043) which is highly resistant to the antifolic acid compound, amethopterin, was gently ruptured by exposing protoplasts of the organism to a hypotonic solution. The crude lysate resulting there-from was treated by various chemical and physical techniques designed to separate folic acid reductase from dihydrofolic acid reductase. In the process, the enzyme was purified approximately 160-fold; however, throughout the process, the enzyme preparation maintained the ability to reduce folic acid to tetrahydrofolic acid. Attempts to isolate mutants showing a deficiency in either folic acid reductase or dihydrofolic acid reductase were unsuccessful. Based on these results, it is concluded that folic acid is reduced to tetrahydrofolic acid by one enzyme in S. faecium (ATCC 8043). The crude lysate was also subjected to ultracentrifugation. An analysis of the supernatant fluid and the sediment indicated that the reductive activity is located in the soluble fraction of the cell.     

Folate polyglutamates in T4D bacteriophage and T4D-infected Escherichia coli

The folate compound which is a structural component of the Escherichia coli T-even bacteriophage baseplates, has been identified as the hexaglutamyl form of folic acid using a new chromatographic procedure (Baugh, C.M., Braverman, E. and Nair, M.G. (1974) Biochemistry 13, 4952–4957). It has also been found that the host cell contains a variety of polyglutamyl forms of folic acid. The major form is the triglutamate (about 50%) but small amounts of higher molecular weightsfolates including the octaglutamate (1.8%) have been identified. Upon infection with wild-type T4D bacteriophage there is a shift in the distribution of the folate compounds so that the folyl polyglutamyl compounds having the higher molecular weights are increased. Infection of E. coli with baseplate mutants of T4D containing an amber mutation in gene 28 resulted in the formation of significant amounts (over 7%) of folate compound(s) of molecular weight much higher than those observed either in uninfected cells or cells infected with wild-type T4D. It is suggested that the T4D gene 28 product functions to cleave glutamate residues from high molecular weight folyl polyglutamates to increase the availability of the folyl hexaglutamate for virus assembly.     

Folic Acid Acts Through DNA Methyltransferases to Induce the Differentiation of Neural Stem Cells into Neurons

The present study investigated the roles of folic acid and DNA methyltransferases (DNMTs) in the differentiation of neural stem cells (NSCs). Neonatal rat NSCs were grown in suspended neurosphere cultures and identified by their expression of SOX2 protein and capacity for self-renewal. Then NSCs were assigned to five treatment groups for cell differentiation: control (folic acid-free differentiation medium), low folic acid (8 μg/mL), high folic acid (32 μg/mL), low folic acid and DNMT inhibitor zebularine (8 μg/mL folic acid and 150 nmol/mL zebularine), and high folic acid and zebularine (32 μg/mL folic acid and 150 nmol/mL zebularine). After 6 days of cell differentiation, immunocytochemistry and western blot analyses were performed to identify neurons by β-tubulin III protein expression and astrocytes by GFAP expression. We observed that folic acid increased DNMT activity which may be regulated by the cellular S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), and the abundance of neurons but decreased the number of astrocytes. Zebularine blocked these effects of folic acid. In conclusion, folic acid acts through elevation of DNMT activity to increase neuronal differentiation and decrease astrocytic differentiation in NSCs.     

饲料中添加叶酸和VB12对中华绒螯蟹幼蟹生长、非特异性免疫和抗病力的影响

为研究叶酸和VB12协同作用对中华绒螯蟹(Eriocheir sinensis)幼蟹生长、非特异性免疫和抗病力的影响,选取初始体重为(2.570.03) g的幼蟹600只,随机分成4组,每组5个重复,每个重复30只幼蟹,分别投喂对照组(不添加叶酸和VB12),单一VB12组(0.2 mg/kg),单一叶酸组(2.3 mg/kg)和联合处理组(0.2 mg/kg VB12 +2.3 mg/kg叶酸)的饲料8周。在养殖实验结束后,先统计成活率和称重,然后从每个处理组随机选取30只幼蟹,用2108 CFU/mL的嗜水气单胞菌注射攻毒2周。实验结果表明:幼蟹的增重率、特定生长率、饲料效率和存活率在联合处理组最高,显著高于对照组(P0.05),但与单一叶酸或VB12组相比不存在显著差异(P0.05)。联合处理组的血清酚氧化酶活性显著高于对照组(P0.05),但与单一叶酸或VB12组也无显著性差异(P0.05)。同时,联合处理组的血清酸性磷酸酶、碱性磷酸酶、溶菌酶活性和血细胞总数等指标最高,其次是单一叶酸组和VB12组,而对照组最低。投喂联合处理组饲料幼蟹的肝胰腺超氧化物歧化酶活性最高,而丙二醛含量和累积死亡率最低。以上结果表明,叶酸和VB12对幼蟹的生长、生理代谢和免疫性能均可能有互补和协同作用,养殖生产中建议饲料中叶酸和VB12添加量分别为2.3 mg/kg和0.2 mg/kg。       

Periconceptional Maternal Folic Acid Use of 400 μg per Day Is Related to Increased Methylation of the IGF2 Gene in the Very Young Child

Background

Countries worldwide recommend women planning pregnancy to use daily 400 µg of synthetic folic acid in the periconceptional period to prevent birth defects in children. The underlying mechanisms of this preventive effect are not clear, however, epigenetic modulation of growth processes by folic acid is hypothesized. Here, we investigated whether periconceptional maternal folic acid use and markers of global DNA methylation potential (S-adenosylmethionine and S-adenosylhomocysteine blood levels) in mothers and children affect methylation of the insulin-like growth factor 2 gene differentially methylation region (IGF2 DMR) in the child. Moreover, we tested whether the methylation of the IGF2 DMR was independently associated with birth weight.

Methodology/Principal Findings

IGF2 DMR methylation in 120 children aged 17 months (SD 0.3) of whom 86 mothers had used and 34 had not used folic acid periconceptionally were studied. Methylation was measured of 5 CpG dinucleotides covering the DMR using a mass spectrometry-based method. Children of mother who used folic acid had a 4.5% higher methylation of the IGF2 DMR than children who were not exposed to folic acid (49.5% vs. 47.4%; p = 0.014). IGF2 DMR methylation of the children also was associated with the S-adenosylmethionine blood level of the mother but not of the child (+1.7% methylation per SD S-adenosylmethionine; p = 0.037). Finally, we observed an inverse independent association between IGF2 DMR methylation and birth weight (−1.7% methylation per SD birthweight; p = 0.034).

Conclusions

Periconceptional folic acid use is associated with epigenetic changes in IGF2 in the child that may affect intrauterine programming of growth and development with consequences for health and disease throughout life. These results indicate plasticity of IGF2 methylation by periconceptional folic acid use.     

Folic Acid Supplementation Promotes Mammary Tumor Progression in a Rat Model

Folic acid supplementation may prevent the development of cancer in normal tissues but may promote the progression of established (pre)neoplastic lesions. However, whether or not folic acid supplementation can promote the progression of established (pre)neoplastic mammary lesions is unknown. This is a critically important issue because breast cancer patients and survivors in North America are likely exposed to high levels of folic acid owing to folic acid fortification and widespread supplemental use after cancer diagnosis. We investigated whether folic acid supplementation can promote the progression of established mammary tumors. Female Sprague-Dawley rats were placed on a control diet and mammary tumors were initiated with 7,12-dimethylbenza[a]anthracene at puberty. When the sentinel tumor reached a predefined size, rats were randomized to receive a diet containing the control, 2.5x, 4x, or 5x supplemental levels of folic acid for up to 12 weeks. The sentinel mammary tumor growth was monitored weekly. At necropsy, the sentinel and all other mammary tumors were analyzed histologically. The effect of folic acid supplementation on the expression of proteins involved in proliferation, apoptosis, and mammary tumorigenesis was determined in representative sentinel adenocarcinomas. Although no clear dose-response relationship was observed, folic acid supplementation significantly promoted the progression of the sentinel mammary tumors and was associated with significantly higher sentinel mammary tumor weight and volume compared with the control diet. Furthermore, folic acid supplementation was associated with significantly higher weight and volume of all mammary tumors. The most significant and consistent mammary tumor-promoting effect was observed with the 2.5x supplemental level of folic acid. Folic acid supplementation was also associated with an increased expression of BAX, PARP, and HER2. Our data suggest that folic acid supplementation may promote the progression of established mammary tumors. The potential tumor-promoting effect of folic acid supplementation in breast cancer patients and survivors needs further clarification.     

2-Deaminofolic acid elicits desensitization without excitation of the cyclic GMP response in Dictyostelium discoideum

Folic acid induces a rapid transient increase in cellular cGMP levels in D. discoideum. A half-maximal cGMP response is effected by about 0.2 μM folate. In addition, extracellular folate is rapidly inactivated by deaminases. The main product 2-deaminofolic acid generates no measurable cGMP response at concentrations of up to 0.1 mM. At 1 mM a 10% response is observed. In contrast, 1 mM deaminofolic acid inhibits the cGMP accumulation as induced by 0.3 μM folic acid by 90%. This antagonism by deaminofolate is competitive with a K_I value of 20 μM. When the cells are preincubated with deaminofolic acid before stimulation with folic acid, the inhibition changes to a non-competitive type (K_I = 20 μM). It is concluded that although deaminofolate does not elicit cGMP accumulation, some cellular process is activated resulting in a diminished cGMP response to saturating folate stimuli. This process of desensitization is characterized by a first-order rate constant of 0.04 s⁻¹, while folic acid-induced desensitization proceeds with a higher rate of 0.6 s⁻¹. We discuss that the latter rate constant is more likely to reflect the rate of detection of a desensitizing signal, rather than the rate of the desensitization process itself.     

Growth of Chaetomium cellulolyticum on Alkali-Pretreated Hardwood Sawdust Solids and Pretreatment Liquor

The treatment of a hardwood sawdust with 1% NaOH solution at 121°C dissolved 19.7% of the dry matter, mainly hemicellulose and lignin. Fermentation of the treated solids by Chaetomium cellulolyticum for 48 h gave a product containing 12.5% crude protein (total N × 6.25) on a dry weight basis. The in vitro rumen digestibility of the 48-h fermentation product was 30%, compared to 24% for the alkali-treated but unfermented sawdust. Growth was independent of sawdust particle size in the range 40 to 100 mesh. Fermentation of the pretreatment liquor gave a product containing up to 50% crude protein (dry weight basis) with an in vitro rumen digestibility of 65 to 76%. Approximately 6.7 g of crude protein was obtained from the treated solids and 2.2 g from the pretreatment liquor per 100 g of sawdust treated. The product from the pretreatment liquor fermentation has potential as a high-protein animal feed supplement but could not be produced economically without an outlet for the relatively indigestible product from the solids fermentation. Growth on the pretreatment liquor was strongly pH dependent; there was a considerable increase in the lag phase when the pH was lowered from 7.5 to 5.2. This effect appears to be due to an inhibitor whose toxicity is reduced at high pH.     

Identification of a folate binder in hog kidney.

A macromolecular binder of folic acid (pteroylglutamic acid) and folic acid derivatives has been identified in extracts of hog kidney. With partially purified preparations, binding of [3H]pteroylglutamate was competed for by unlabeled pteroylglutamate, 5-methyltetrahydrofolic acid and its triglutamate derivative, by tetra- and dihydrofolic acid, and by N-10-formyltetrahydrofolic acid. The partially purified extract did not bine [3H]methotrexate nor could methotrexate or 5-formyltetrahydrofolic acid compete for [3H]folic acid-binding sites. The rate of binding of pterolyglutamate at 37 degrees was approximately 3%/s, was independent of pteroylglutamate concentration, and was essentially irreversible between pH 6.0 and 9.0. Below pH 6.0 binding was reversible, and at pH 3.5 the folic acid-binder complex completely disassociated. Based upon Sephadex gel filtration, the molecular weight of the folate-binder complex is 35,000 to 40,000. Binding activity was unaffected by pretreatment with ribonuclease or deoxyribonuclease but was completely destroyed by trypsin. The initial, unfractionated extract showed gamma-glutamyl carboxypeptidase (conjugase) activity which was lost in subsequent steps of purification of the folate binder.     

Folic Acid Prevents Behavioral Impairment and Na+,K+-ATPase Inhibition Caused by Neonatal Hypoxia–Ischemia

Folic acid plays an important role in neuroplasticity and acts as a neuroprotective agent, as observed in experimental brain ischemia studies. The aim of this study was to investigate the effects of folic acid on locomotor activity, aversive memory and Na(+),K(+)-ATPase activity in the frontal cortex and striatum in animals subjected to neonatal hypoxia-ischemia (HI). Wistar rats of both sexes at postnatal day 7 underwent HI procedure and were treated with intraperitoneal injections of folic acid (0.011 μmol/g body weight) once a day, until the 30th postnatal day. Starting on the day after, behavioral assessment was run in the open field and in the inhibitory avoidance task. Animals were sacrificed by decapitation 24 h after testing and striatum and frontal cortex were dissected out for Na(+),K(+)-ATPase activity analysis. Results show anxiogenic effect in the open field and an impairment of aversive memory in the inhibitory avoidance test in HI rats; folic acid treatment prevented both behavioral effects. A decreased Na(+),K(+)-ATPase activity in striatum, both ipsilateral and contralateral to ischemia, was identified after HI; a total recovery was observed in animals treated with folic acid. A partial recovery of Na(+),K(+)-ATPase activity was yet seen in frontal cortex of HI animals receiving folic acid supplementation. Presented results support that folic acid treatment prevents memory deficit and anxiety-like behavior, as well as prevents Na(+),K(+)-ATPase inhibition in the striatum and frontal cortex caused by neonatal hypoxia-ischemia.     

Radioiodinated pteroyltyrosine: A novel analog for folate radioassay

Pteroyltyrosine, a folate analog was synthesized by the condensation of 6-formylpterin and p-aminobenzoyl-l-tyrosine methyl ester followed by dimethylamine borane reduction and mild deesterification. The radioiodinated product was employed in a competitive protein binding assay for pteroylglutamic (folic) acid.     

Cytogenetic effects in peripheral blood lymphocytes in descendants of chernobyl disaster liquidators under the impact of mitomycin c in vitro and folic acid in vivo

Data on cytogenetic examinations of the descendans of Chernobyl disaster liquidators (cleanup personnel) have been obtained. It has been established that the level of spontaneous chromosomal aberrations before folic acid administration was 1.8 times higher than that value after its employment (4.45 vs 2.42%, p < 0.01). In lymphocyte cultures treated with mitomycin C accompanied by folic acid, it was 4.5 times higher before their administration (23.95 vs 5.36%, p < 0.001). The data obtained confirm the possibility of stabilizing the genetic apparatus in the descendans of Chernobyl disaster liquidators after folic acid administration.     

Nutrient media for plant-parasitic nematodes. VI. Nucleic acids content and nucleotide synthesis in Aphelenchoides rutgersi

The nucleic acids content of Aphelenchoides rutgersi, Hooper and Myers, was 0.9% DNA and 2.6% RNA dry weight. The DNA contained 29.5% adenine, 29.3% thymine, 22.5% guanine, and 18.8% cytosine, while the RNA was composed of 22.8% adenine, 23.0% uracil, 31.4% guanine, and 22.9% cytosine on a molar basis.The nematodes needed folic acid for reproduction regardless of the presence or absence of nucleic acid supplements in the culture medium. This was shown by including aminopterin, a folic acid antagonist in the culture medium. A 2-hr incubation of nematodes with glycine-¹⁴C (U) and orotic-5-³H acid resulted in the incorporation of ³H-label into both DNA and RNA. Only the RNA fraction contained a significant amount of ¹⁴C-label. When this RNA was fractionated, the adenine and guanine accounted for the ¹⁴C-label, while cytidylic and uridylic acids contained the ³H-label, thereby demonstrating purine and pyrimidine synthesis by A. rutgersi. The incorporation of orotic acid into the pyrimidines was 8 times higher than that of glycine into purines.     

Oxidative and reductive cleavage of folates--a critical appraisal.

A critical analysis has been made of the oxidative and reductive techniques employedfor cleavage of the C⁹-N¹⁰ bond of folic acid and its derivaatives. The assumption has previously been made that these cleavage reactions reduce folates to a common family of p-aminobenzoylglutamate derivatives varying only in the lengths of γ-polyglutamyl peptide side chains which are readily subjected to quantitative and qualitative analysis. This assumption is incorrect. Oxidation by potassium permanganate effectively cleaved folic acid, dihydrofolic acid, tetrahydrofolic acid, and 5-formyltetrahydrofolic acid to yield p-aminobenzoylglutamate. 5-Methyltetrahydrofolic acid was merely oxidized to 5-methyldihydrofolic acid while 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid were oxidized to 10-formylfolate which was stable to further attack. Of all the folate derivatives tested only folic acid and dihydrofolic acid were cleaved to p-aminobenzoylglutamate by the zinc-hydrochloric acid reduction method. Both tetrahydrofolic acid and 5-methyltetrahydrofolic acid were stable under fully reducing conditions. 5,10-Methenyl-,10-formyl-, and 5-formyltetrahydrofolic acid yielded N-methyl-p-aminobenzoylglutamate. It is evident, therefore, that not only is the dominant mammalian tissue folate derivative, 5-methyltetrahydrofolate, resistant to cleavage by either method, but that a common family of p-aminobenzoylglutamate derivatives is not the end product of those folate compounds that are susceptible. While this may not invalidate the reports of the relative polyglutamate chain lengths of tissue folates such data should be regarded with some caution.     

Effect of In Ovo Feeding of Folic Acid on Subsequent Growth Performance and Blood Constituents Levels in Broilers

This study designed to determine effect of in ovo feeding of folic acid on subsequent growth performance and blood constituents levels in broilers. A total of 1000 fertile broiler eggs were divided into four groups. Control group (1) received no injection. In group 2, eggs received in ovo feeding of distiller water (40 µg). Group 3 received in ovo feeding of folic acid (40 µg). Groups 4 and 5 were similar to Group 3, except eggs injected with 80 and 120 µg of folic acid. All eggs were incubated and after hatch chickens were randomly assigned into their experimental groups. On days 1 and 42 post-hatch, chicken randomly selected and blood constituents, carcass characteristics, food intake, body weight gain and food conversion ratio were determined. According to the results, no significant difference detected on hatchability rate of the in ovo injected eggs (P?>?0.05). Dose dependent increase observed in glucose and folic acid levels in chicken in ovo injected with folic acid on day 1 post hatch (P?=?0.001). Blood glucose, folic acid and phosphorous levels increased (P?=?0.001) while cholesterol, triglyceride, HDL and LDL, calcium and alkaline phosphatase decreased in ovo injected with folic acid on day 42 post hatch (P?=?0.001). Food conversion ratio increased by in ovo injection of the folic acid (P?=?0.001). These results suggest folic acid had positive effects in broiler chicken.     

Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4′-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K_m values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4′-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20–30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20–30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.     

Prophylactic and therapeutic effects of Mytilus edulis fatty acids on adjuvant-induced arthritis in male Wistar rats

Lipid-rich fractions from the flesh tissue of Mytilus edulis were obtained by solvent extraction and chromatographic separation, and tested for anti-inflammatory (AI) activity in vitro and in vivo. Inhibition of leukotriene production by isolated human neutrophils in response to calcium ionophore stimulation in the presence of exogenous arachidonic acid substrate was demonstrated for the hydrolysed triglyceride fraction of the crude lipid extract. This fraction was subsequently tested for in vivo AI activity using the mycobacterial adjuvant-induced polyarthritis rat model. The hydrolysed triglyceride fraction showed significant AI activity when dosed therapeutically (10 mg/kg BW/day, p.o., for 6 days from the onset of arthritis), decreasing body weight loss by 55% and hind paw swelling by 65% compared to the arthritic control. The (non-hydrolysed) crude lipid extract was effective when dosed prophylactically (30 mg/kg BW/day, p.o., for 16 days starting on day ?2 of arthritigen inoculation). Structural analysis by GC and GC–MS revealed in the extracts an abundance of EPA (20:5n-3) and DHA (22:6n-3) (37% of total fatty acids), along with a small quantity of a rare anti-inflammatory n-3 analogue of arachidonic acid, namely 7, 11, 14, 17-eicosatetraenoic acid (20:4n-3).     

Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide adenine dinucleotide glycohydrolase, nicotinamide mononucleotide glycohydrolase, and nicotinamide adenine dinucleotide pyrophosphatase activities.

Extracts of Salmonella typhimurium were chromatographed by using Sephadex G-150 to separate the various enzymes involved with pyridine nucleotide cycle metabolism. This procedure revealed a previously unsuspected nicotinamide adenine dinucleotide (NAD) glycohydrolase (EC 3.2.2.5) activity, which was not observed in crude extracts. In contrast to NAd glycohydrolase, NAD pyrophosphatase (EC 3.6.1.22) was readily measured in crude extracts. This enzyme possessed a native molecular weight of 120,000. Other enzymes examined included nicotinamide mononucleotide (NMN) deamidase (EC 3.5.1.00), molecular weight of 43,000; NMN glycohydrolase (EC 3.2.2.14), molecular weight of 67,000; nicotinic acid phosphoribosyl transferase (EC 2.4.2.11), molecular weight of 47,000; and nicotinamide deamidase (EC 3.5.1.19), molecular weight of 35,000. NMN deamidase and NMN glycohydrolase activities were both examined for end product repression by measuring their activities in crude extracts prepared from cells grown with and without 10(-5) M nicotinic acid. No repression was observed with either activity. Both activities were also examined for feedback inhibition by NAD, reduced NAD, and NADP. NMN deamidase was unaffected by any of the compounds tested. NMN glycohydrolase was greatly inhibited by NAD and reduced NAD, whereas NADP was much less effective. Inhibition of NMN glycohydrolase was found to level off at an NAD concentration of ca. 1 mN, the approximate intracellular concentration of NAD.