The effect of exogenously supplied hydroxylamine on glutamate dehydrogenase,nitrate reductase,and nitrite reductase in isolated pea roots

Hydroxylamine added to the nutrient medium in sublethal concentrations (0.2 to 1.0 mN) enhanced NADH₂ dependent glutamate dehydrogenase activity in isolated pea roots. The increase in activity depended on proteosynthesis and was lower in the presence of NO₃ ^? and NH₄ ⁺ ions. The induction of nitrate reductase and of nitrite reductase was partly inhibited by sublethal hydroxylamine concentrations.     

The regulation of glutamate dehydrogenase,nitrite reductase,and nitrate reductase in excised pea roots by nitrite

Both nitrite reductase and nitrate reductase were induced by nitrite, but there were differences in the time course of induction and in the response to different NO₂ ^- concentrations between these enzymes. NH₄ ⁺ depressed the induction of nitrite reductase. NADH₂ dependent glutamate dehydrogenase activity was enhanced by those NO₂-concentrations in the medium at which unmetabolized NO₂ ^- occurred in the roots. NADPH₂ and NAD+ dependent GDh activities were not affected. In vivo modification and (or) in vivo activation were probably responsible for the increase in NADH₂ dependent GDH activity.     

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and F_oF₁-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and F_oF₁-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by F_oF₁-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.     

Biochemical characterization of a metallothionein-like protein in rat brain

Recent investigations from this laboratory have identified a metallothionein-like protein in the rat brain with an elution volume (ve/vo) of 2.08 and a molecular weight of smaller than 10,000. The synthesis of this protein was stimulated following intracerebroventricular (icv, 0.20 μmol zinc/μL/h, 48 h), but not intraperitoneal (ip) administration of ZnSO₄. Furthermore, chronic ip administration of ZnSO₄ (5.0 mg/kg/d/10 d) did not alter the level of the metallothionein-like protein in the brain. However, the hepatic metallothionein was induced following icv administration of ZnSO₄. The chromatofocusing of metallothionein-like protein isolated by gel permeation chromatography on Sephadex G-75 exhibits three zinc-binding peaks, which focus on pH 6.8, 6.2, and 5.3, respectively. It is expected that the protein peak focusing at 5.3 is a metallothionein-like protein. Purification of the zinc stimulated metallothionein-like protein on ion exchange chromatography on DEAE-Sephadex A-25 columns, using a linear gradient elution procedure produced two isoforms, eluting, respectively, at 75 and 137 mM of Tris-acetate buffer, pH 7.5. The comparative high-performance liquid chromatographic (HPLC) profiles of the zinc-induced hepatic metallothionein isoforms I and II (retention times 17.39 and 18.73 min) and that of the zinc-stimulated metallothionein-like protein isoforms I and II (retention times 17.32 and 18.64 min) are very similar. The function(s) of the metallothionein-like protein isoforms in the brain remains to be elucidated.     

Research of Iron Reduction and the Iron Reductase Localization of Anammox Bacteria

The iron-reducing capability of anammox bacteria was examined in this study using Percoll purified anammox bacteria. Anammox bacteria could reduce Fe(III) to Fe(II) with organic matters as the electron donor. The activity of anammox iron-reducing process was dependent on different electron donor, acceptor and pH. The highest iron-reducing activity of anammox bacteria was achieved with Fe(III)-NTA (nitrilotriacetic acid) as electron acceptor and formate as the electron donor at pH7. Similar to other iron reducers, 80 % of the iron reductase in anammox bacteria was located in the membrane fraction. Due to the chemical oxidant of NO₂ ^? and the NO₃ ^? dependent ferrous iron oxidation by anammox bacteria, the iron-reducing activity of anammox bacteria could be severely inhibited when iron-reducing pathway and the anammox process were coupled. However, the total nitrogen removal efficiency was not significantly affected in the presence of Fe(III). The iron-reducing capability of anammox bacteria could influence both N and Fe cycle on earth, and it is a potential way for wastewater treatment.     

Inhibition and uncoupling of the ADP-regulated electron transport in isolated chloroplasts

The effects of energy transfer inhibitors, electron transport inhibitors and uncouplers on the ADP-regulated and ADP-independent activity of ferricyanide reduction in isolated spinach chloroplasts were studied. Phlorizin and sulfate did not affect the ADP-independent ferricyanide reduction. In the ADP-regulated reduction, these reagents did not affect the ADP inhibition process but inhibited the activity restoration process due to phosphorylation. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and linolenic acid depressed both ADP-regulated and ADP-independent activity of ferricyanide reduction. Gramicidin S and 2-amino-1-butanol depressed ADP-regulated activity and stimulated ADP-independent activity. The decrease in the ADP-regulated ferricyanide-reducing activity (restoration) due to (incomplete) uncoupling paralleled the decrease in phosphorylation activity (P/Δe=1).     

The action of exogenous gibberellic acid on polysome formation and translation of mRNA in germinating castor-bean seeds

The amount of protein synthesis in germinating castor-bean seeds has been estimated by the quantitative and qualitative exmainatin of polysomes from the seeds in the presence and absence of gibberellic acid (GA₃). Careful optimisation of polysome extraction procedures was required to minimise the ribonuclease activity in the extracts. Ribonuclease activity in seed extracts increased fourfold over the first 5 d of germination. Gibberellic acid stimulated polysome formation about twofold during the first 4 d of germination. It also stimulated the amount of mRNA associated with polysomes by about twofold during the first 3 d of germination. Between days 1 and 5 of germination, polysome formation was primarily limited by mRNA availability. During the period 0–24 h, polysome formation was independent of mRNA levles. The increase in enzyme activities stimulated by GA₃ was probably the result of an increase in the amount of cellular mRNA. No evidence was obtained for an action of GA₃ on translation other than on the increased production of RNA. Examination of the recruitment of isocitrate-lyase mRNA into polysomes showed that GA₃ did not specifically stimulate production of this enzyme.     

Purification and Characterization of Two Soluble C1−-Activated Arginyl Aminopeptidases from Human Brain and Their Endopeptidase Action on Neuropeptides

Two closely related Cl(-)-activated arginyl aminopeptidases (I and II) were purified from a soluble extract of postmortem human cerebral cortex by anion-exchange chromatography and preparative gel electrophoresis. The electrophoretic mobility of II was approximately 80% that of I; the molecular mass of both enzymes was approximately 70 kilodaltons (kDa) (gel filtration). The aminopeptidase action of I and II on aminoacyl-7-amido-4-methylcoumarin (AMC) substrates was restricted to the Arg and Lys derivatives. Both enzymes had significant endopeptidase activity, hydrolysing several biologically active peptides including neurotensin, bradykinin, angiotensin-I, substance P, luliberin, and somatostatin at internal bonds. Other peptides [Leu-enkephalin, proctolin, thyroliberin, adrenocorticotropin18-39 (ACTH18-39), ACTH11-24, and dynorphin (1-13)] were not appreciably hydrolysed. The amino- and endopeptidase activities had pH optima at 6.5 and 7, respectively, and were both inhibited by metal ion chelators and sulphydryl group blocking agents. The aminopeptidase activity was stimulated 20-fold by Cl- ions, whereas the endopeptidase activity was unaffected by the latter. Km values for neurotensin degradation were 20 microM (I) and 37 microM (II) and for Arg-AMC hydrolysis they were 167 microM (I) and 125 microM (II). The endopeptidase activity was not inhibited by the aminopeptidase inhibitors arphamenine or bestatin (IC50 = 9 nM and 0.1 microM, respectively, with Arg-AMC substrate).     

The inhibitory effect of angiotensin II on BKCa channels in podocytes via oxidative stress

Angiotensin II (Ang II) is an important active substance of the renin-angiotensin system (RAS). The present study has confirmed that abnormalities of Ang II may be related with cerebrovascular diseases, endocrine diseases, cardiovascular diseases, liver diseases, such as: cerebral hypoxia, diabetes, obesity, atrial fibrillation, and liver cirrhosis. However, understanding effects of Ang II on podocytes is not enough. This study was to investigate the effects of oxidative stress on the large conductance, Ca²⁺-activated K⁺ channels (BK_Ca). Results from the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay showed that Ang II induced podocyte death in a concentration-dependent manner. The measurement of superoxide dismutase (SOD) generation demonstrated that Ang II decreased the total SOD of cellular levels. Meaningfully, pretreatment of a type of ROS scavenger formulations named N-(mercaptopropionyl)-glycine (N-MPG) could inhibit podocyte apoptosis induced by Ang II. Meanwhile, patch-clamp technique was used in this study to detect the effects of Ang II on currents of BK_Ca channel in podocytes. The results indicated that Ang II inhibited the current amplitude of BK_Ca channel and decreased the slope of I–V curve. Ang II also made the activation curves of BK_Ca channel shift to the left. These results may provide a theoretical basis for potential treatment of chronic glomerular disease in the future.     

Estimates of heavy metal tolerance and chromium(VI) reducing ability of Pseudomonas aeruginosa CCTCC AB93066: chromium(VI) toxicity and environmental parameters optimization

The potential role of parameters in the reduction of hexavalent chromium [Cr(VI)] by Pseudomonas aeruginosa is not well documented. In this study, laboratory batch studies were conducted to assess the effect of a variety of factors, e.g., carbon sources, salinity, initial Cr(VI) concentrations, co-existing ions and a metabolic inhibitor, on microbial Cr(VI) reduction to Cr(III) by P. aeruginosa AB93066. Strain AB93066 tolerated up to 400 mg/L of Cr(VI) in nutrient broth medium compared to only 150 mg/L of Cr(VI) in nutrient agar. This bacteria exhibited different levels of resistance against Pb(II) (200 mg/L), Cd(II) (100 mg/L), Ni(II) (100 mg/L), Cu(II) (100 mg/L), Co(II) (50 mg/L) and Hg(II) (5 mg/L). Cr(VI) reduction was significantly promoted by the addition of glucose and glycerine but was strongly inhibited by the presence of methanol and phenol. The rate of Cr(VI) reduction increased with increasing concentrations of Cr(VI) and then decreased at higher concentrations. The presence of Ni(II) stimulated Cr(VI) reduction, while Pb(II), Co(II) and Cd(II) had adverse impact on reduction ability of this strain. Cr(VI) reduction was also inhibited by high levels of NaCl, various concentrations of sodium azide and 20 mM of SO₄ ^2?, MoO₄ ^2?, NO₃ ^?, PO₄ ^3?. No significant relationship was observed between Cr(VI) reduction and redox potential of the culture medium. Scanning electron microscopy showed visible morphological changes in the cells due to chromate stress. Fourier transform infrared spectroscopy analysis revealed chromium species was likely to form complexes with certain functional groups such as carboxyl and amino groups on the surface of P. aeruginosa AB93066. Overall, above results are beneficial to the bioremediation of chromate-polluted industrial wastewaters.     

Purification and characterization of a class II α-Mannosidase from Moringa oleifera seed kernels

α-Mannosidase (EC. 3.2.1.114) belonging to class II glycosyl hydrolase family 38 was purified from Moringa oleifera seeds to apparent homogeneity by conventional protein purification methods followed by affinity chromatography on Con A Sepharose and size exclusion chromatography. The purified enzyme is a glycoprotein with 9.3 % carbohydrate and exhibited a native molecular mass of 240 kDa, comprising two heterogeneous subunits with molecular masses of 66 kDa (α-larger subunit) and 55 kDa (β-smaller subunit). Among both the subunits only larger subunit stained for carbohydrate with periodic acid Schiff’s staining. The optimum temperature and pH for purified enzyme was 50 °C and pH 5.0, respectively. The enzyme was stable within the pH range of 3.0–7.0. The enzyme was inhibited by EDTA, Hg²⁺, Ag²⁺, and Cu²⁺. The activity lost by EDTA was completely regained by addition of Zn²⁺. The purified enzyme was characterized in terms of the kinetic parameters K _m (1.6 mM) and V _max (2.2 U/mg) using para-nitrophenyl-α-D-mannopyranoside as substrate. The enzyme was very strongly inhibited by swainsonine (SW) at 1 μM concentration a class II α-Mannosidase inhibitor, but not by deoxymannojirimycin (DMNJ). Chemical modification studies revealed involvement of tryptophan at active site. The inhibition by SW and requirement of the Zn²⁺ as a metal ion suggested that the enzyme belongs to class II α-Mannosidase.     

Purification and properties of thiosulfate reductase from Desulfovibrio gigas

Thiosulfate reductase of the dissimilatory sulfate-reducing bacterium Desulfovibrio gigas has been purified 415-fold and its properties investigated. The enzyme was unstable during the different steps of purification as well as during storage at-15°C. The molecular weight of thiosulfate reductase estimated from the chromatographic behaviour of the enzyme on Sephadex G-200 was close to 220 000. The absorption spectrum of the purified enzyme exhibited a protein peak at 278 nm without characteristic features in the visible region. Thiosulfate reductase catalyzed the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate, and exhibited tetrathionate reductase activity. It did not show sulfite reductase activity. The optimum pH of thiosulfate reduction occurred between pH 7.4 and 8.0 and its K _m value for thiosulfate was calculated to be 5·10^-4 M. The sensitivity of thiosulfate reductase to sulfhydryl reagent and the reversal of the inhibition by cysteine indicated that one or more sulfhydryl groups were involved in the catalytic activity. The study of electron transport between hydrogenase and thiosulfate reductase showed that the most efficient coupling was obtained with a system containing cytochromes c ₃ (M _r =13000) and c ₃ (M _r =26000).     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

Fumarate reduction inProteus mirabilis

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorateresistant mutant strains of the wild type, fumarate reductase appeared to be affected.
2. Cytoplasmic membrane suspensions isolated from anaerobically grownP. mirabilis oxidized formate and NADH with oxygen and with fumarate, too.
3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochromeb, cytochromea ₁ and cytochromed. Cytochromeb was reduced by NADH as well as by formate to approximately 80%.
4. 2-n-Heptyl-4-hydroxyquinoline-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state.
5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochromeb.
6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen.
7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochromeb as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grownP. mirabilis is presented.

     

A monoclonal antibody-based enzyme-linked immunosorbent assay of aflatoxin B1 in peanut products

An improved enzyme-linked immunosorbent assay (ELISA) combined with monoclonal antibody (MAb) and one-step extraction method was established for the estimation of aflatoxin B₁ (AFB₁) in a peanut product. AFB₁ was converted to AFB₁-oxime, and then conjugated with bovine serum albumin (BSA). Spleen cells from mice immunized with AFB₁-BSA conjugates were fused with myeloma cells. After double selection with AFB₁-ovalbumin (OVA) and carbodiimide-modified OVA, five stable hybridoma cells secreting anti-AFB₁ MAbs (AF1, AF 2, AF 3, AF 4, and AF5) were cloned. Using these anti-AFB₁ MAbs, we developed the indirect competitive ELISA (cELISA) with alkaline phosphatase (ALP) — labeled sheep anti — mouse IgG as marker and the direct cELISA with AFBi-oxime horseradish peroxidase (POD) as marker. The minimum detectable limits of the indirect cELISA with AF 1, 2, 3, 4, and 5 were 5, 5, 5, 5, and 50 pg of standard AFB₁ per assay, respectively, and those of the direct cELISA with AF 1, 3, 4, and 5 were 2.5, 5, 25, and 100 pg of standard AFB₁ per assay, respectively. The cross reactivity of each toxins with these MAbs in the indirect cELISA was as follows: (a) AF 1 and AF 2 were reactive with AFB₂ as well as AFB₁, weakly with AFG₂ > AFG₁ > aflatoxicol II (COL II) > aflatoxicol I (COL I) and less weakly with other aflatoxins; (b) AF 3 and AF 4 were reactive with COL II as well as AFB₁, weakly with COLI > AFQ₁ and less weakly with others; (c) AF 5 was AFQ₁ as well as AFB₁ weakly with COL II > AFG₂ > COL I and less weakly with others. The 60% aqueous methanol extracts of oil-roasted blanched peanuts (“butter peanut”), naturally contaminated with AFB₁, were assayed by the direct cELISA without further purification. The direct cELISA with the most sensitive MAb AF 1 was allowed to determine 1 ng of AFB₁ per g samples.     

The electron transport system of the anaerobic Propionibacterium shermanii

1. Electron transport particles obtained from cellfree extracts of Propionibacterium shermanii by centrifugation at 105000xg for 3 hrs oxidized NADH, d,l-lactate, l-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too.
2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome a ₁ and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80–90.
3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or “site I region”, in the electron transport system of P. shermanii.
4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor, and reached 80–100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase.
5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions.
6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations of the inhibitor. Inhibitors of terminal oxidases were inactive, except for carbon monoxide.

     

Identification and properties of UDP-glucose: Cyanidin-3-O-glucosyltransferase isolated from petals of the red campion (Silene dioica)

An enzyme catalyzing the transfer of the glucosyl moiety of UDP-glucose to the 3-hydroxyl group of cyanidin has been demonstrated in petal extracts of Silene dioica mutants with cyanidin-3-O-glucoside in the petals. This transferase activity was also present in young rosette leaves and calyces of these plants. The highest glucosyltransferase activity was found in petals of opening flowers of young plants. The enzyme was purified ninetyfold by PVP and Sephadex chromatography. The glucosyltransferase had a pH optimum of 7.5, had a “true K_m value” of 4.1×10^?4 m for UDP-glucose and 0.4×10^?4 m for cyanidin chloride, and was not stimulated by divalent metal ions. Both p-chloromercuribenzoate and HgCl₂ inhibited the enzyme activity. Pelargonidin chloride and delphinidin chloride at reduced rates also served as substrates. The enzyme did not catalyze the glucosylation of the 3-hydroxyl group of flavonols or the 5-hydroxyl group of anthocyanins. ADP-glucose could not serve as a glucosyl donor. The results of Sephadex G150 chromatography suggest that the glucosyltransferase can exist as dimer of about 125,000 daltons and as active monomers of 60,000 daltons. The genetic control of the glucosyltransferase activity is discussed.     

Naphthalene oxygenase fromCorynebacterium renale: Characterisation and mechanism of oxygenation

The formation ofcis-l,2,-dihydroxy-l,2,-dihydronaphthalene from naphthalene by naphthalene oxygenase, purified fromCorynebacterium renale ATCC 15075, was demonstrated to involve oxidation of a mol NADH and consumption of one mol oxygen. The enzyme contains one g-atom Fe²⁺ and one FAD. Catalase inhibited product formation and H2O2 could substitute for NADH in the reaction. Superoxide dismutase inhibited enzyme activity when either NADH or H₂O₂ was present; the generation of superoxide anion on addition of NADH to the enzyme, in the absence of naphthalene, was detected by the nitro blue tetrazolium reduction method. Hydroxyl radical scavengers, ethanol, mannitol and sodium benzoate, inhibited product formation when either NADH or H₂O₂ was present. Electron spin resonance studies, under aerobic conditions, indicated that iron of the enzyme underwent valence changes during the course of the reaction     

Effect of some nitrogenous salts on nitrogen transfer and protease activity in germinatingZea mays L. seeds

Maize seeds were allowed to germinate in the presence of different nitrogenous salts for 72 h. Changes in the ethanol soluble and insoluble nitrogen were studied in the embryo and in the endosperm. Supply of Ca(NC₃)₂ enhanced germination and protease activity in the endosperm resulting in greater solubilisation of protein to soluble nitrogen in the seeds. NH₄NO₃ and (NH₄)₂SO₄ were less effective as compared to Ca(NO₃)₂. Cycloheximide inhibited germination and protease activity. Pretreatment also resulted in increase in growth, soluble and insoluble nitrogen, and nitrate reductase activity in the primary leaves. Ca(NO₃)₂ was more effective than NH₄NO₃ and (NH₄)₂SO₄.