Differential expression of glutathione reductase and cytosolic glutathione peroxidase,GPX1, in developing rat lungs and kidneys

A mutant rat GPX1 (a cytosolic predominant form), in which the selenocysteine residue in the catalytic center was replaced by cysteine, was prepared and an antibody against the mutant enzyme was raised. The resultant antibody specifically reacted with rat GPX1 and was, together with the Glutathione reductase (GR) antibody, used in a Western blot analysis and immunohistochemistry experiments. To elucidate the physiological coupling of these enzymes under oxidative stress which accompanies the birth, developmental changes of the protein levels and enzymatic activities of GR and GPX1 were examined for lungs and kidneys from prenatal fetus to adult rats. The expression of GR was already evident at the prenatal stage and remained high in lungs at all stages. However, GR activity in kidneys gradually increased after birth reaching maximal levels at adulthood. An immunohistochemical study showed that GR was strongly bound to the bronchial epithelia in lungs and the epithelial cells of renal tubes. GPX1 was expressed in the renal tube epithelial cells and its level gradually increased after birth in a manner similar to that of GR. The expression of GPX1 in the lungs was, on the other hand, variable and occurred in some alveolar cells and bronchial epithelia only at restricted periods. It preferentially localized in nuclei at a late stage of development. Thus, the expression of the two functionally coupled enzymes via GSH did not appear to coordinate with development, tissue localization or under oxidative stress. Since many gene products show GSH-dependent preoxidase activity, other peroxidase(s) may be induced to compensate for the low GPX1 levels at stages with high GR expression.     

Human pancreas protein 2 (PAN2) has a retinal reductase activity and is ubiquitously expressed in human tissues

Human gene for pancreas protein 2 (PAN2) is a novel member of the short-chain dehydrogenase/reductase gene superfamily. The properties of PAN2 protein have not yet been characterized. We present the first evidence that human PAN2 is a ubiquitously expressed microsomal enzyme that recognizes retinoids but not steroids as substrates with the apparent K(m) values between 0.08 microM and 0.4 microM. PAN2 is approximately 4-fold more efficient in the reductive than in the oxidative direction. The apparent K(m) values for NADP(+) and NADPH are 0.65 microM and 0.32 microM versus 1200 microM and 1060 microM for NAD(+) and NADH, respectively. Kinetic constants and expression pattern of PAN2 suggest that it is likely to function as a reductase in vivo and might contribute to the reduction of retinaldehyde to retinol in most human tissues.     

Effects of β-naphthoflavone on the cytochrome P450 system,and phase II enzymes in gilthead seabream (Sparus aurata)

The effect of β-naphthoflavone (β-NF) on several catalytic activities of cytochrome P450 (CYP) and phase II enzymes putatively controlled by [Ah]-receptor activation in the liver, heart and kidney of gilthead seabream, was investigated. In the liver, β-NF treatment [intraperitoneal injection (i.p.) 50 mg/kg] resulted in an increase of CYP content, immunoreactive CYP 1A and methoxyresorufin-O-demethylase (MEROD), pentoxyresorufin O-depentylase (PROD) and ethoxyresorufin-O-deethylase (EROD) activities. However, β-NF had no effect on any of the hepatic phase II enzymes examined (benzaldehyde dehydrogenase, propionaldehyde dehydrogenase, glutathione S-transferase, UDP-glucuronyl-transferase, DT-diaphorase). Single i.p. injection of 10 mg/kg β-NF showed a maximal induction of CYP 1A-like protein and EROD activity after 3–7 days. CYP 1A and EROD returned to control levels 18-days post-treatment. β-NF injection also caused a rapid increase of a single band size of mRNA recognized by a CYP 1A1 cDNA fragment from sea bass (Dicentrarchus labrax). Expression of mRNA preceded the increase of EROD activity and declined rapidly by 96 h. Dose–response experiments demonstrated that EROD was significantly enhanced in liver by a single injection of 0.3 mg/kg β-NF and was the most sensitive measurement for CYP 1A-like induction. β-NF treatments also increased the expression of CYP 1A-like protein, mRNA and EROD, but not MEROD and PROD activities in heart and kidney.     

Microbiological activities of nucleotide loop-modified analogues of vitamin B12

Novel vitamin B₁₂ analogues in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analogue containing imidazole instead of 5,6-dimethylbenzimidazole as well as cobinamide methyl phosphate were tested for biological activities with Escherichia coli 215, a B₁₂- or methionine-auxotroph, and Lactobacillus leichmannii ATCC 7830 as test organisms. A cyano form of 5,6-dimethylbenzimidazolyl tetramethylene, trimethylene and hexamethylene analogues supported the growth of L. leichmannii in this order. 5.6-Dimethylbenzimidazolyl dimethylene and imidazolyl trimethylene analogues did not show B₁₂ activity and behaved as weak B₁₂ antagonists when added together with cyanocobalamin. An adenosyl form of the biologically active analogues served as coenzymes for ribonucleotide reductase of this bacterium, whereas that of the inactive analogues did not. The latter acted as weak competitive inhibitors against adenosylcobalamin. ON the contrary, all the analogues did not support the growth of E. coli 215 at all by themselves and inhibited the growth when added with a suboptimum level of cyanocobalamin. A methyl form of the analogues also did not support the growth of E. coli 215, although they served as active coenzymes for methionine synthase of the bacterium. Since unlabeled analogues strongly inhibited the uptake of [³H]cyanocobalamin by this bacterium, it seems likely that the analogues exert their anti-B₁₂ activity toward E. coli 215 by blocking the B₁₂-transport systemAbbreviations AdoCbl adenosylcobalamin - MeCbl methylcobalamin - CN-Cbl cyanocobalamin or vitamin B₁₂ - Cbl cobalamin - (CN, aq)Cbi cyanoaquacobinamide - MeCbi methylcobinamide - Cbi cobinamide - (CN, aq)Cbi-PMe cyanoaquacobinamide methyl phosphate - Cbi-PMe cobinamide methyl phosphate - DBI 5,6-dimethylbenzimidazole - DBIyl 5,6-dimethylbenzimidazolyl - FMNH₂ fully reduced form of riboflavin 5-phosphate     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Purification and Characterization of Cytochrome P450 Reductase from Liver Microsomes of Feral Leaping Mullet (Liza saliens)

NADPH-cytochrome P450 reductase was purified to electrophoretic homogeneity from detergent-solubilized liver microsomes from the leaping mullet (Liza saliens). The purified reductase was characterized with respect to spectral, electrophoretic, and biocatalytic properties. In addition, effects of pH, ionic strength, and the substrate concentration on the NADPH-dependent cytochrome c reductase activity of the purified fish liver cytochrome P450 reductase were studied. Cytochrome P450 reductase was purified 438-fold with a yield of 17.5% with respect to the initial amount present in the fish liver microsomes. The specific activity of the enzyme was found to be 52.6 μmol cytochrome c reduced per minute per mg protein. The monomer molecular weight of the purified enzyme was calculated to be 77,000 ± 1000 when electrophoresed on polyacrylamide gels under the denaturing conditions in the presence of SDS. The absorption spectrum of fish reductase showed two peaks at 378 and 455 nm. NADPH-dependent cytochrome c reductase activity of the purified Liza saliens liver cytochrome P450 reductase was found to be maximal when pH was between 7.4 and 7.8. The apparent K_m of the purified enzyme was found to be 7.69 μM for cytochrome c when the enzyme activity was measured in 0.3 M potassium phosphate buffer, pH 7.7, at room temperature, and the enzyme was fully saturated by its substrate, cytochrome c, when the substrate concentration was at or above the 70 μM. Furthermore, the purified enzyme was biocatalytically active in reconstituting the 7-ethoxyresorufin O-deethylase activity in the reconstituted system containing purified mullet liver cytochrome P4501A1 and lipid. These results suggested that the purified fish liver cytochrome P450 reductase is similar to its mammalian counterparts with respect to spectral, electrophoretic, and biocatalytic properties. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 12: 103–113, 1998     

Haemonchus contortus: molecular cloning, sequencing, and expression analysis of the gene coding for the small subunit of ribonucleotide reductase

Gastro-intestinal (GI) parasites are of great agricultural importance, annually costing the livestock industry vast amounts in resources to control parasitism. One such GI parasite, Haemonchus contortus, is principally pathogenic to sheep; with the parasite's blood-feeding behaviour causing effects ranging from mild anaemia to mortality in young animals. Current means of control, which are dependent on repeated treatment with anthelmintic chemicals, have led to increasing drug resistance. Together with the growing concern over residual chemicals in the environment and food chain, this has led to attempts to better understand the biology of the parasite with the aim to develop alternate or supplementary means of control, including the development of molecular vaccines. As a first step towards the understanding of the synthesis of deoxyribonucleotides in H. contortus, and its potential application to therapeutic control of this economically important parasite, we report the cloning, sequencing, and mRNA expression analysis of the ribonucleotide reductase R2 gene.     

N+离子注入长豇豆对硝酸还原酶及氮代谢的影响

不同剂量的Ｎ＾＋离子注入长豇豆种子。豇豆开花结荚后，低剂量Ｎ＾＋离子处理能增加叶片的ＮＲ活性，促进ＮＯ＾－３－Ｎ向氨基酸和蛋白质的转化；减少ＮＯ＾－３－Ｎ的积累，提高叶片蛋白质和氨基酸含量。高剂量Ｎ＾＋离子处理抑制官种转化，豇豆结荚后期，叶片ＮＯ＾－３－Ｎ积累增多。     

Ribonucleotide Reductases: Radical Chemistry and Inhibition at the Active Site

Abstract

Ribonucleoside 5′-diphosphate reductases (RDPRs) have been studied for several decades. Increasingly sophisticated mechanisms have been proposed for the reduction of natural substrate ribonucleotides to their 2′-deoxy counterparts and for mechanism-based inactivation of RDPRs with 2′-substituted-ribonucleotides. We now discuss biomimetic reactions of model substrate and inhibitor analogues, which clarify three aspects of previously proposed mechanisms postulated to occur at the active site of RDPRs.     

Surface Charges and Regulation of FMN to Heme Electron Transfer in Nitric-oxide Synthase

The nitric-oxide synthases (NOS, EC 1.14.13.39) are modular enzymes containing attached flavoprotein and heme (NOSoxy) domains. To generate nitric oxide (NO), the NOS FMN subdomain must interact with the NOSoxy domain to deliver electrons to the heme for O₂ activation during catalysis. The molecular basis and how the interaction is regulated is unclear. We explored the role of eight positively charged residues that create an electropositive patch on NOSoxy in enabling the electron transfer by incorporating mutations that neutralized or reversed their individual charges. Stopped-flow and steady-state experiments revealed that individual charges at Lys⁴²³, Lys⁶²⁰, and Lys⁶⁶⁰ were the most important in enabling heme reduction in nNOS. Charge reversal was more disruptive than neutralization in all cases, and the effects on heme reduction were not due to a weakening in the thermodynamic driving force for heme reduction. Mutant NO synthesis activities displayed a complex pattern that could be simulated by a global model for NOS catalysis. This analysis revealed that the mutations impact the NO synthesis activity only through their effects on heme reduction rates. We conclude that heme reduction and NO synthesis in nNOS is enabled by electrostatic interactions involving Lys⁴²³, Lys⁶²⁰, and Lys⁶⁶⁰, which form a triad of positive charges on the NOSoxy surface. A simulated docking study reveals how electrostatic interactions of this triad can enable an FMN-NOSoxy interaction that is productive for electron transfer.