The homologous tryptophan critical for cytochrome c peroxidase function is not essential for ascorbate peroxidase activity

The crystal structures of ascorbate peroxidase (APX) and cytochrome c peroxidase (CCP) show that the active site structures are nearly identical. Both enzymes contain a His-Asp-Trp catalytic triad in the proximal pocket. The proximal Asp residue hydrogen bonds with both the His proximal heme ligand and the indole ring nitrogen of the proximal Trp. The Trp is stacked parallel to and in contact with the proximal His ligand. This Trp is known to be the site of free radical formation in CCP compound I and also is essential for activity. However, APX forms a porphyrin radical and not a Trp-centered radical, even though the His-Asp-Trp triad structure is the same in both peroxidases. We found that conversion of the proximal Trp to Phe has no effect on APX enzyme activity and that the mutant crystal structure shows that changes in the structure are confined to the site of mutation. This indicates that the paths of electron transfer in CCP and APX are distinctly different. The Trp-to-Phe mutant does alter the stability of the APX compound I porphyrin radical, by a factor of two. Electrostatic calculations and modeling studies show that a potassium cation located about 8?Å from the proximal Trp in APX, but absent in CCP, makes a significant contribution to the stability of a cation Trp radical. This underscores the importance of long-range electrostatic effects in enzyme catalyzed reactions.     

小麦返白系与不同基因型小麦品种杂交后代IPO表达的研究

以小麦返白系和对照矮变１号以及返白系与各不同生态类型的冬性、半冬性、春性小麦的杂交、回交Ｆ１、Ｆ２代为材料,研究这些不同基因型品种在返白期间过氧化物酶同工酶（ＩＰＯ）基因表达模式的动态变化特点。结果表明,在返白期间以返白系为母本的各杂交、回交品种的白化苗中,ＩＰＯ的个别酶分子表现了可逆的基因阻遏和去阻遏的表达现象。这种表达特点在正、反杂交后Ｆ１代中表现一致,从遗传模式上分别属于质－核互作型的分子遗     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Ethylene effects on peroxidases and cell growth patterns in Picea abies hypocotyl cuttings

The effect of 2-chloroethylphosphonic acid (ethrel) on cell growth patterns and per-oxidase activity (EC 1.11.1.7) and location in young Norway spruce cuttings ( Picea abies [L.] Karst.) was investigated. The peroxidase activity in a fraction containing soluble and membrane bound enzymes show a diurnal variation, with decreased activity during the light period and a corresponding increase during the following dark period. The decrease during the day could to some extent be counteracted by treatment with ethrel. It appears that ethrel affects only peroxidases in the isolated membrane fraction, since peroxidases bound to the cell wall were not affected by ethrel. In vitro experiments indicated that the hydrophobicity of soluble peroxidases was increased by treatment with ethylene. Cytochemical localization of peroxidase activity in differentiating tracheids revealed a clear ethrel-induced increase in the tonoplast. It appears that ethylene affects soluble peroxidases in vivo in such a way that they are directed to a more hydrophobic environment, like the tonoplast. Treatment with ethrel also changed the appearance of the rough endoplasmic reticulum (ER) and Golgi apparatus. Dilated ER cisternae were observed on electron micrographs, as a result of treatment with ethrel. The number of vesicles produced by the Golgi apparatus and also the amount of vesicles fusing with the plasma membrane in secondary-wall-forming tracheids increased considerably. The results clearly indicate that the stimulatory effect of ethylene in spruce seedlings on lignification and cell wall formation, is due to a general stimulation on both synthesis, transport and secretion of cell wall material and not on a stimulation of peroxidase activity as reported for other species.     

Effects of moderate drought on ascorbate peroxidase and glutathione reductase activities in mesophyll and bundle sheath cells of maize

Mesophyll and bundle sheath cells of maize leaves ( Zea mays L.) both contain the enzymes ascorbate peroxidase (AP; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) which are involved in hydrogen peroxide detoxification. Since bundle sheath cells of maize are deficient in photosystem II and have high CO₂ levels, oxidative stress may be less severe in these cells than in mesophyll cells. The present study was conducted to determine if AP and GR activity levels preferentially increase in mesophyll cells relative to bundle sheath cells when plants are subjected to moderate drought. Although drought inhibited the growth of greenhouse-grown plants, it did not affect the levels of protein, chlorophyll or AP. GR was unaffected by drought in whole leaf tissue and mesophyll cells, but did increase slightly in bundle sheath cells. This slight increase is of questionable biological importance. AP and GR activity levels were similar in mesophyll cells, bundle sheath cells and in whole leaf tissue. The data suggest that moderate drought has little effect on enzymes of the hydrogen peroxide scavenging system and that mesophyll and bundle sheath cells may be exposed to similar levels of hydrogen peroxide.     

Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.     

Generation of anNco I restriction site for translational fusions using PCR-mediated,site-directed mutagenesis

A polymerase chain reaction-based method of site-directed mutagenesis was used to introduce anNco I restriction site on the translation start site of a tomato peroxidase gene. This quick and efficient method utilized two overlapping synthetic oligonucleotide primers containing the requisite base pair changes on the ATG translation start site and two flanking primers in PCR. The resulting DNA amplified fragments were fused together byNco I digestion at the mutated ends followed by a T4 ligation reaction. A rapid alternative method utilizing the overlapping fragments and the flanking primers in PCR can also be used for ligating the two fragments. Cloning and sequencing of the PCR-amplified fragments provided additional evidence for the presence of the site-specific mutations. Unique restriction sites upstream and downstream of the site-specific mutation allows for the easy transfer of this mutated region into the wild type peroxidase gene.     

Influence of carbon and nitrogen sources on glutathione catabolic enzymes inCandida albicans during dimorphism

The effect of carbon sources, glucose and sucrose, and nitrogen sources such as ammonia, glutamate andl-citrulline on the activities of glutathione metabolic enzymes has been studied. Yeast and mycelial cells were used to identify changes in activity levels of glutathione reductase (GSSGR), glutathione transferase (GST), glutathione peroxidase (GPX) and -glutamyl transpeptidase (GGT). Enzyme activities from cells grown in sucrose media were lower than in glucose media regardless of the enzyme tested, morphological form, or the growth interval. In all enzymes except GST, activity was higher in yeast form than in mycelia, regardless of nitrogen source, with lower activity from 24 to 72 h than at 96 h. In citrulline media, yeast form showed the maximum GST, GGT, and GPX activity. In ammonia-amended media, mycelia showed maximum activity in GGT, whereas in glutamate media, mycelia showed the maximum activity in GST. Also, the type of nitrogen source had no effect on GPX activity in the mycelial form. Finally, changing the nitrogen source showed no significant effect on GSSGR activity, either in the yeast or mycelial form.     

秦艽体细胞胚不同发育阶段几种同工酶的特性

以秦艽（ＧｅｎｔｉａｎａｃｒａｓｓｉｃａｕｌｉｓＤｕｔｈｉｅｅｘＢｕｒｋｉｌｌ）无菌苗幼嫩茎叶为外植体,在ＭＳ培养基上诱导出胚性愈伤组织及体细胞胚。本文比较了秦荒体细胞胚发育过程中几种酶的活性及同工酶变化。过氧化物酶与细胞色素氧化酶均在球形胚阶段出现一活性高峰;酯酶同工酶在体细胞胚发育过程中伴有特征带Ｅ＿２、Ｅ＿６的出现;酸性磷酸酯酶的活性与体细胞胚发育程度呈正相关。所以认为只要综合运用这几种同工酶的实验数据,就可以灵敏检测体细胞胚的发育进程。     

NALL-1, an acute lymphoblastic leukemia cell line with peroxidase activity

Summary All cells examined from the non-B, non-T acute lymphoblastic leukemia cell line, NALL-1, stained positive both for terminal deoxynucleotidyl transferase and for common ALL antigen. In addition, peroxidase activity was detected by light microscopy in 55 to 75% of cells and peroxidase-positive granules were detected ultrastructurally in >80% of cells. Peroxidase activity in NALL-1 may result from derepression of peroxidase genes or clonal proliferation of a biphenotypic precursor cell.