Godson O. Osuji

N-CARBOXYMETHYLCHITOSAN INHIBITION OF AFLATOXIN PRODUCTION : ROLE OF ZINC

Aqueous Solutions of N-carboxymethylchitosan (NCMC) suppressed both growth and aflatoxin production byAspergillusflavus andA.parasiticus in submerged culture (Adye and Mateles A&M). Test media were amended with various concentrations of zinc (15, 30, 45, 60 uM), and NCMC solution (0.62 uM). After 8 days incubation NCMC-treated cultures showed marked reduction of aflatoxin production and fungal growth. Enhanced levels of zinc did not overcome the NCMC-mediated inhibition of fungal growth or aflatoxin production.

Ammonium ion salvage by glutamate dehydrogenase during defence response in maize

Abstract When phytochemical defence response was elicited by treatment of maize with chitosan, the binomial isoenzymes of l -glutamate dehydrogenase (GDH, EC 1.4.1.2) isomerized. The amination electrode potential of the GDH charge isomers decreased by 10 mV in the chitosan-treated maize. GDH isomerization and amination are therefore spontaneous reactions. The free l -Glu and l -Gln were 3.84 and 3.68 μmol, respectively, per 10 g of the chitosan-treated maize. The amination activities of GDH showed that the 10 mV decrease in electrode potential accounted fully for the 3.84 μmol free l -Glu content of the chitosan-treated maize. Therefore the binomial GDH synthesized all the l -Glu, thereby salvaging at least 51% of the NH 4 + liberated during phytochemical defence response, while glutamine synthetase salvaged the remaining NH 4 + . The chitosan-elicited isomerization of maize GDH and the accompanying 10 mV decrease in electrode potential widened the plant-pathogen amination energy barrier, thereby minimizing the amount of NH 4 + the pathogen can absorb from the plant.

Purification of Glutamate Dehydrogenase Isoenzymes and Characterization of Their Substrate Specificities

Abstract Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, α, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06–0.8 mM of each of UTP, ATP, GTP, and CTP, 0–100.0 mM NH4Cl, 0–50.0 mM α-ketoglutaratr (α-KG), 0–0.2 mM NADH, 0–10.0 mM CaCl2 5 units of DNase 1, antibiotics, and ∼5 µg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16°C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and α-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for the isoenzymes in RNA synthesis reaction. The product RNA was up to 2 µg µg−1 GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [α-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

Regulation of peanut glutamate dehydrogenase by methionine sulphoximine

Peanut glutamate dehydrogenase (GDH) was electrophoretically purified to homogeneity. Rotofor IEF fractionated the peanut GDH to 7 isoelectric (charge) isomers, which focused in the pH 5-8 range. Western blot analysis of the charge isomers using anti-GDH serum showed that methionine sulphoximine (MSX) treatment suppressed the b-subunit (69 KDa), but enhanced the a-subunit (45 KDa), and alpha-subunit (46 KDa) of the enzyme. The MSX-mediated suppression of the b-subunit increased the NH4+ Km value of the acidic charge isomers from 7.7 mM in the control to 50 mM in the MSX-treated peanut, and also increased the 2-ketoglutarate Km value of the basic charge isomers from 0.4 mM in the control to 7.0 mM in the MSX treatment. Therefore, the control peanut could salvage NH4+ with its GDH activity. But by increasing the NH4+ Km value, the MSX rendered the enzyme ineffective in NH4+ salvage. In the deamination direction, despite the enhancement of the a-, and alpha-subunits by MSX, the basic charge isomers of GDH had very high Km value for L-glu (50 mM), similar to that (25 mM) of the control. Thus, the GDHs of the control, and MSX treatment could not function in the deamination reaction in vivo. These results show that the treatment of peanut with MSX impaired the amination function of GDH.

Discovery of the RNA Synthetic Activity of Glutamate Dehydrogenase andIts Application in Drug Metabolism Research

Glutamate dehydrogenase (GHD) synthesizes some RNAs that regulate mRNA abundance in response to the environment. The connection of gene expression and drug metabolism by the GDH-synthesized RNA has not been dem- onstrated experimentally. The regulation of the mRNAs encoding the drug-metabolizing enzymes was studied by northern hybridization using the GDH-synthesized RNAs as probes. The mRNAs encoding cytochrome P-450 reductase, UDP- glucosyltransferase, alternative oxidase, and ABC-transporters were upregulated by the administered ATP+UTP+GTP. Also superoxide dismutase and GSH S-transferase were upregulated by administered ATP. The untreated control, GTP, and UTP did not upregulate any of the mRNAs. The mRNAs encoding the enzymes were coordinately regulated at the molecular level. All the enzymes are also active in drug detoxication in mammals. Photometric assays of enzyme activi- ties confirmed that the enzymes were present at levels proportional to their respective encoding mRNAs as detected by the GDH-synthesized RNA probes. Genetic code-based nucleic acid probes were partially accurate in detecting the mRNAs encoding the enzymes. Therefore, GDH-synthesized RNAs are important genetic metabolic probes for the screening of mRNAs encoding the drug metabolizing enzymes. Nucleoside triphosphates and analogs are antihypertensives, antineo- plastics, antiarrhythmics, antimetabolites, antiviral agents etc and they induce GDH isomerization.

Biomass Enhancement in Maize and Soybean in Response to Glutamate Dehydrogenase Isomerization

The relationship between nutrient composition, crop biomass, and glutamate dehydrogenase (GDH) isoenzyme pattern was investigated in soybean (Glycine max) and maize (Zea mays) by monitoring the nutrient induced isomerization of the enzyme from the seedling stage to the mature crop. GDH was extracted from the leaves of the plants, and the isoenzymes were fractionated by isoelectric focusing followed by native polyacrylamide gel electrophoresis. The isomerization Vmax values for soybean GDH, similar to maize GDH increased curvilinearly from 200 – 400 μmol mg−1 min−1 as the inorganic phosphate nutrient applied to the soil decreased from 50 − 0 mM. In soybean, combinations of N and K, P, or S nutrients induced the acidic and neutral isoenzymes, and gave biomass increases 25 – 50 % higher than the control plant. GDH isoenzymes were suppressed in soybean that received nutrients without N, K, or P and accordingly the biomass was about 30 % lower than the control. Treatment of maize with NPK nutrients increased the GDH Vmax values from 138.9 at the vegetative to 256.4 μmol mg−1 min−1 at the reproductive phase, and suppressed the basic isoenzymes, but induced both the acidic and neutral isoenzymes thereby inducing seed production (27.0 ± 1.4 g per plant); whereas both the acidic and basic isoenzymes were suppressed in the control maize, and seeds did not develop. Simultaneous induction of the acidic, neutral, and basic isoenzymes of GDH indicated the occurrence of senescence. Therefore in maize and soybean, the induction of the acidic and basic isoenzymes of GDH led to the enhancement of biomass.

Nucleotide-Dependent Reprogramming of mRNAs Encoding Acetyl Coenzyme A Carboxylase and Lipoxygenase in Relation to the Fat Contents of Peanut

The nucleotide-dependent reprogramming of the mRNAs encoding acetyl coenzyme A carboxylase (ACC, EC 6.4.1.2) and lipoxygenase (LO, EC 1.13.11.12) was studied by Northern analysis. Equal concentrations of total RNA from the peanut (Arachis hypogaea L. cv. “Valencia”) seedlings were probed with those glutamate dehydrogenase (GDH)-synthesized RNAs that were homologous to the mRNAs encoding ACC and LO. Treatment (4NTPs solution) of the peanuts increased the abundance of the mRNAs encoding the LO and ACC about 5 and 10 folds respectively but decreased the fat accumulation by 37% compared with the control peanut. Treatments (pyrimidine NTPs)of the peanut induced up to 60% more fat accumulation than the control. The high fat (40.0–44.0%) contents of the UTP-, and 3NTP-treated peanuts were characterized by very high abundance of the mRNA for ACC, but low abundance of the mRNA for LO. The inverse relationship between the abundance of the mRNAs encoding ACC and LO compared with the seedlings fat contents was due to the plus/minus strands sequence homologies between the Northern probes for ACC and LO. Therefore, LO, ACC, and the GDH-synthesized RNAs participated in the regulation of the fat contents. These results could be useful in the environmental manipulation of the quantity and quality of peanut oil.

The isomerization of glutamate dehydrogenase in response to lead toxicity in maize

Maize (Zea mays) was cultivated on lead-adultrated soil up to 600 mg(Pb) kg-1. At maturity, the maize seeds were harvested. The glutamate dehydrogenase (GDH) was fractionated to its isoenzyme population by Rotofor isoelectric focusing (IEF). The increasing Pb concentration progressively enhanced the more acidic isoenzymes (pI 6.3 - 6.5), and at the same time suppressed the less acidic isoenzymes (pI 7.3 - 7.8) and at the 600 mg(Pb) kg-1(soil) only the most acidic couple of isoenzymes (pI 6.3, and 6.5) were detectable. The NH4+ Km values of the GDH increased progressively from 6.2 in the control to 100 mM and the total glutathione content of maize seeds from 60 to 240 nmol g-1 in the 600 mg(Pb) kg-1(soil) treated maize. The orderly, and sequential isomerization of GDH in response to Pb suggests that the enzyme functions as a sensor in the monitoring of environmentally induced stress.

Peanut glutamate dehydrogenase: A target site of herbicide action

Germination of peanut seeds in 5 mM N-(phosphonomethyl) glycine isopropylamide, 1, 2-dihydro-3, 6-pyridazinedione (maleic hydrazide), glyphosate, and isopropylamine solutions induced stress response reactions and the release of 24–100 µmol NH4 + per 50 g of seed, with consequent differential suppression of the subunits of glutamate dehydrogenase (GDH). Changes in the GDH isomerization suggested an interaction of the herbicides with the enzyme with consequent increases in the Michaelis constants (Km) from 7.7 mM in the control to 45.0, 50.0 and 77.0 mM NH4 + in the glyphosate, maleic hydrazide, and glyphosate isopropylamide treatments respectively.

N‐Carboxymethylchitosan enhancement of the storage protein contents of maize seeds (Zea mays L.)

Abstract Maize plants growing in the field and maize seeds germinating in the greenhouse were treated with 10mM N‐carboxy‐methylchitosan (NCMC) solutions. The field‐grown NCMC‐treated maize seeds had a higher zein content (6.3%) than control maize seeds (3.0%). Two temperatures (25°C and 35°C) were used for seed germination. At 35°C, the NCMC‐treated seedlings had a higher zein content (5.6%) than the control seedlings (1.8%). Also the NCMC‐treated seedlings had a higher total cellular RNA content (20.6 μg/g) than the control seedlings (11.0 μg/g). Therefore NCMC doubled both the zein and the cellular RNA contents of the maize.