Urease from leaves of Glycine max and Zea mays

Extracts of Glycine max and Zea mays leaves catalysed the release of ¹⁴CO₂ from [¹⁴C] urea with multiple pH maxima (5.5 and 9.0 for G. max; 5.5, 7.5 and 8.8 for Z. mays). Evidence was obtained that the principal activities, at pH 5.5 and 8.8–9.0 catalysed the same reaction stoichiometry as did urease purified from jackbean seeds (EC 3.5.1.5). The urease activities with these pH optima were not resolved by ammonium sulphate fractionation, DEAE-cellulose chromatography, or gel filtration chromatography. Many structural analogues of urea inhibited leaf urease, the most effective being amino acid hydroxamates, hydroxyurea and selenourea. Allantoic acid and ureidoglycolate are probably not alternative substrates because they showed at most only weak competitive inhibition with respect to radioactive urea.     

细菌脲酶蛋白复合物UreABC的表达与纯化

细菌脲酶能分解尿素为氨,在瘤胃尿素氮代谢中发挥重要作用.为了表达纯化并研究蛋白脲酶复合物UreABC,通过PCR扩增脲酶基因簇的结构基因ureA、ureB、ureC,将ureB构建在含有N端His标签的pet28a+载体,ureA、ureC基因共同构建在含有N端His标签的表达载体pETDuet-1,经酶切及测序鉴定得...     

Alcaloides du Camoensia maxima

Three alkaloids, leontidine, camoensine and camoensidine, the two latter being new, have been extracted from Camoensia maxima. A partial synthesis     

Purification and properties of carbonic anhydrase from Chlamydomonas reinhardii

Carbonic anhydrase (CA) was purified from the unicellular green alga Chlamydomonas reinhardii, and the purity of the preparation was established by gradient gel electrophoresis. The purified enzyme exhibited a MW of 165 000 and contained 6 atoms of Zn. The subunit MW, as determined by dodecyl sulfate electrophoresis, was 27 000. These results are consistent with a quarternary structure which is hexameric, each monomer containing 1 g atom of Zn. Like spinach CA, and in contrast to other oligomeric plant CAs, a sulfhydryl reducing agent is not needed to stabilize the enzyme. CO₂-hydrase activity was inhibited by both acetazolamide (I₅₀ = 7.8 × 10^?9M) and sulfanilamide (I₅₀ = 1.3 × 10^?5M), as well as by certain inorganic anions. The purified enzyme showed relatively weak esterase activity with p-nitrophenyl acetate but was an extremely effective esterase with 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone as the substrate. Both esterase activities could be completely inhibited by adding acetazolamide. In its gross structural characteristics, the C. reinhardii enzyme resembles the CAs from higher plants. However, in its esterase activity and the inhibition by sulfonamides it is markedly different from plant CAs and bears more resemblance to erythrocyte CAs.     

Ornithine transaminase from Cucurbita maxima cotyledons

During germination a marked increase in both soluble and particulate ornthine transaminase occurs in pumpkin cotyledons. Both enzymes had a pH optimum of 8.3 and a requirement for ornthine and α-ketoglutarate. Other keto acids or amino donors showed little activity. The enzymes required an active sulphydryl group for maximum activity. Exogenous pyridoxal phosphate was not required, but hydroxylamine inhibited the reaction and added pyridoxal phosphate overcame this inhibition. Proline inhibited the reaction and may play a role in the fate of ornithine in pumpkin cotyledons.     

重组幽门螺杆菌ureA,ureB低温表达、纯化及抗原鉴定

目的：建立经济有效的方法,从大肠杆菌表达,纯化重组幽门螺杆菌尿素酶A、B亚单位。方法：含重组表达质粒pGEX-2T/ureA,pGEX-2T/ureB大肠杆菌分别在不同浓度IPTG、不同温度条件下作诱导培养,以SDS-PAGE分析表达产物,采用谷胱甘肽-Sepharose 4B亲和层分离纯化表达产物,以Western-blotting和ELISA对纯化产物作抗原性分析鉴定。结果：IPTG诱导浓度为0.1mmol/L;诱导温度为28℃,含重组质粒大肠杆菌表达可溶性融合蛋白GST-ureA、GST-ureB各约占总表达产物的78%和87%,经亲和层析,得纯度90%以上的GST-ureA约为14mg/L。GST-ureB约为18mg/L;融合蛋白呈ureA、ureB抗原性反应。结论：建立了低温诱导及纯化高纯度ureA、ureB基因表达产物的方法,所得纯化产物具有ureA、ureB抗原活性,为进一步的应用研究打下基础。     

Urease and urea amidolyase: Determination of activity in liverworts

Using highly sensitive techniques, we have investigated urea degradation in the liverworts and found that they have high urease but no detectable urea amidolyase activity.     

Polysaccharide from cell walls of Chlamydomonas reinhardtii

The cell wall fraction of the green alga Chlamydomonas reinhardtii contained about 25% carbohydrate after prolonged treatment with salivary amylase     

Nine isoflavones from Tephrosia maxima

The isolation and characterisation of nine closely related 3′,4′-disubstituted isoflavones from the aerial parts and roots of Tephrosia maxima     

Flavodoxin from the blue-green alga Nostoc strain MAC

A flavodoxin was isolated from the blue-green alga Nostoc strain MAC grown photoautotrophically or chemoheterotrophically in iron-deficient medium. In vitro, the flavodoxin would support NADP⁺ photoreduction by photosynthetic membranes, pyruvate oxidation by the phosphoroclastic system of Clostridium pasteurianum, and electron transfer to Cl. pasteurianum hydrogenase. In its oxidized form, the flavodoxin had absorbance maxima at 274 sh283 sh293, 376 sh432 and 466 sh488 nm. Reduction by dithionite proceeded via a neutral, blue semiquinone radical. The flavodoxin contained 1 mol of FMN per mol of protein and the amino acid composition showed a predominance of acidic residues; cysteine was apparently absent. A minimum MW of ca 22 000 derived from these data was confirmed by electrophoresis on SDS-polyacrylamide gels and by ultracentrifugal analysis. This flavodoxin thus belongs to the higher MW group of these low potential electron transfer proteins.     

Isolation and properties of a ferredoxin from Anabaena Flos-Aquae

Ferredoxin was isolated from the blue-green alga Anabaena flos-aquae. Its homogeneity was shown by conventional and SDS-polyacrylamide gel electrophoresis, and isoelectric focusing on polyacrylamide gel columns, the latter indicating a pI at ca pH 3·7. The absorption spectrum had, in the oxidized state, maxima at 462, 421, 327 and 276 nm, with a shoulder at 284 nm, a spectrum characteristic of plant-type ferredoxins. The 421 : 276 nm absorbance ratio was typically 0.49. The ferredoxin effectively mediated the photoreduction of NADP⁺ by barley chloroplasts depleted of native ferredoxin. The MW obtained by sedimentation-equilibrium and sedimentation velocity-diffusion coefficient studies was ca 12 000 daltons, a value somewhat higher than suggested by amino acid composition data. The ferredoxin contained 2Fe and 2S per molecule.     

交联脲酶聚集体的制备和初步应用

为提高游离脲酶的稳定性,将游离脲酶用硫酸铵沉淀下来后,以戊二醛作为交联剂对其进行化学交联,制备新型的固定化脲酶交联脲酶聚集体,并对其酶学性质进行研究。交联脲酶聚集体的最适pH、最适温度和K_m值分别为：pH 8.0、70℃和0.021 mol/L。在对交联脲酶聚集体的热稳定性,储存稳定性,和对抗蛋白水解酶的能力的研究中,交联脲酶聚集体均显示了比游离脲酶更高的稳定性。为考察其使用效果和稳定性,将其与包醛氧淀粉联合,用于慢性肾衰动物模型的口服治疗。以腺嘌呤灌胃法(每天300mg/kg, 共30d)制备慢性肾衰动物模型,将23只大鼠随机分为模型对照组(每天以10mL/kg蒸馏水灌胃)、单纯包醛氧淀粉组(给予含包醛氧淀粉饲料,10mL/kg蒸馏水灌胃)和包醛氧淀粉+交联脲酶聚集体组(给予含包醛氧淀粉饲料,交联脲酶聚集体悬浮液10mL/kg灌胃),经2周治疗后, 模型对照组、治疗对照组和治疗组实验前后的肌酐含量均有小幅下降,但差异不显著(P值分别为0.922、0.972和0.225＞0.05)。模型对照组的尿素氮含量变化不明显(P=0.211＞0.05)。治疗对照组和治疗组实验前后的尿素氮含量均明显下降(P值分别为0.004和小于0.001,均小于0.01)。治疗对照组和治疗组实验前后的尿素氮含量下降量比较,有显著性差异(P=0.016＜0.05)。治疗组尿素氮含量下降更明显。说明交联脲酶聚集体和包醛氧淀粉联合使用时,对尿素的清除效率比单纯使用包醛氧淀粉更高。     

De novo synthesis and hormonal regulation of a dipeptidase in Cucurbita maxima

The following evidence was obtained for the de novo synthesis of dipeptidase in squash (Cucurbita maxima Duch. var. Hubbard) cotyledons during germination: (i) the amount of [¹⁴C]leucine incorporated into the dipeptidase was greater than that found in other proteins; (ii) the enzyme coincided with a peak of radioactivity in DEAE column chromatography; and (iii) the specific radioactivity of the enzyme increased with purification. There was also a positive correlation between the rate of [¹⁴C]leucine incorporation into dipeptidase and the rate of dipeptidase development. Four plant growth regulators, gibberellic acid (GA) benzyladenine (BA), indol-3-acetic acid (IAA), and abscisic acid (ABA) were examined for their effect on the development of dipeptidase activity at 5 × 10^?6 and 5 × 10^?5 M. None of these regulators affected the activity of the isolated dipeptidase per se. In intact see ds, BA and IAA inhibited the development of dipeptidase activity at the higher concentration, ABA reduced the activity at both concentrations; however, GA enhanced its development at the higher concentration. In distal-half cotyledons, BA and GA stimulated enzyme development but they showed no synergistic effect. IAA suppressed the development of enzyme activity at the higher concentration and ABA inhibited development at both levels.     

Bromo-compounds of the red alga Lenormandia prolifera

Six bromo-compounds and one bromo-chloro-compound have been detected in Lenormandia prolifera (C.Ag.) J. Agardh (Amansieae; Rhodomelaceae). Hydrolysis of the red pigment floridorubin from the same alga yielded five bromo-, one bromo-chloro and one chloro-phenol. The two main phenols of floridorubin were 2,3-dibromo-4,5-dihydroxy benzyl alcohol (lanosol) and 3,5-dibromo-p-hydroxybenzyl alcohol.     

α-galactosidase of Poterioochromonas malhamensis

In cell-free extracts of Poterioochromonas malhamensis, α-galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) was stable at pH 8. The pH optimum of the enzyme was 7. The enzyme was purified 10-fold through chromatographic steps involving DEAE-cellulose, hydroxylapatite and Sephadex G-200. The apparent MW was 360 000 by sucrose density-gradient centrifugation. All activity was lost on subjecting the enzyme to polyacrylamide-gel electrophoresis. The substrate specificity of the enzyme was examined and some kinetic values determined. The enzyme displayed an unusual activity curve with respect to isofloridoside.     

Peroxidase from the green alga Enteromorpha linza

An enzyme displaying peroxidase activity has been extracted and purified 27-fold from the green alga Enteromorpha linza. The partially purified pre     

Peroxidase activity in the brown alga Laminaria digitata

Cell-free extracts of the brown alga Laminaria digitata catalyse the oxidation of o-dianisidine and of iodide, as well as the formation of iodoamino acids. The enzyme(s) requires hydrogen peroxide for these activities, which are strongly inhibited by cyanide and azide. It is suggested that the activity may be due to a haem-containing peroxidase which, in extracts, is strongly bound, possibly to alginate.     

Isotactic polymethoxy-1-alkenes from the blue-green alga Tolypothrix conglutinata var. Chlorata

4(S*),6(S*),8(S*),10(S*),12(R*),14(R*),16(R*),18(R*),20(R*)-Nonamethoxy-1-pentacosene and smaller amounts of the isotactic homologs, 4,6,8,10,12,14,16,18,20,22-decamethoxy-1-heptacosene and 4,6,8,10,12,14,16,18-octamethoxy-1-tricosene, are novel lipophilic constituents of the toxic blue-green alga Tolypothrix conglutinata var. chlorata Ghose from Fanning Atoll.     

Effects of anoxia and flooding on alcohol dehydrogenase in roots of Glyceria maxima and Pisum sativum

Flood tolerant Glyceria maxima and intolerant Pisum sativum were compared in respect of the effects of anoxia and flooding on the maximum catalytic activities of alcohol dehydrogenase in their roots. Small (<73%) increases in enzyme activity occurred when excised roots of both species were incubated in nitrogen for up to 2 days. Further incubation in nitrogen rapidly and permanently damaged the roots of both species. Enzyme activity in flooded roots of Glyceria was about double that in corresponding non-flooded roots. A marginally greater difference was found for roots of Pisum. It was concluded that the two species respond so similarly to the above treatments that variation in the extent of induction of alcohol dehydrogenase is unlikely to be a significant factor in determining their ability to tolerate flooding.     

幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺的研究

通过不同培养基、不同葡萄糖浓度、不同溶氧条件、补料与非补料对幽门螺杆菌尿素酶B亚单位(UreB)基因工程菌的菌体生长与外源蛋白表达量的影响的比较 ,建立了稳定、适宜的幽门螺杆菌尿素酶B亚单位基因工程菌发酵工艺。多批实验结果证明 ,菌体单产可达 86 g/L ,目的蛋白的表达率为 38.2 %。