磷酸缓冲液对小麦苗保护酶系的影响及与SO2污染的关系

磷酸缓冲液对保护酶系的影响因处理方式和小麦 (Triticum aestivum L.)品种而异。一般是 SOD和 POD活性显著增强 ,前者随 SO2 熏气剂量的增加而降低 ,但较对照为高 ,并有新同工酶谱产生 ;后者随 SO2 熏气剂量的增加而增高 ,并与磷酸缓冲液呈交互促进效应 ,同工酶谱显著增强。作为清除自由基保护酶系的变化 ,可能是磷酸缓冲液减轻小麦苗 SO2 伤害的原因之一。     

The Protective Effect of Phosphate Buffer on SO2 Harmed Wheat Seedling

When 1/15 mol/L of phosphate buffer at pH 6. 4, was used to spray wheat seedling three times, the plasmolemma structure become stabilized and k+ efflux was decreased. After fumigation with SO2 the K+ efflux of wheat seedling ,sprayed with phosphate buffer, decreased markedly. At SO2 dosage of 1.54 ppm × 4h, the decreasing rate of K+ efflux was 54. 11%–81.29% and the decrease rate was in concert with the increase of SO2 dosage. Therefore, the authors consider that the phosphate buffer, at pH 6.4, has a good protective effect against SO2 insult.     

二氧化硫对小麦的氧化胁迫及其某些信号分子的调节

通过在密闭的培养箱中一次性通入不同体积浓度的SO₂气体,研究了小麦幼苗超氧自由基O2-含量和3种抗氧化酶活性的变化,探讨了信号分子水杨酸、乙烯和过氧化氢对SO₂氧化胁迫的调节作用.结果表明,当通入10和40 μl·L^-1 SO₂时,小麦叶片中O2-含量递增,过氧化物酶(POD)和过氧化氢酶(CAT)活性增强,但超氧化物歧化酶(SOD)活性降低.当SO₂浓度达到50 μl·L^-1时,POD和CAT活性也开始降低,此时叶片尖端出现坏死,叶片绿色部位滋生大量真菌.用1 mmol·L^-1水杨酸(SA)(pH6.5)浸泡小麦干种子6 h,或用10 mmol·L^-1 H₂O₂浸泡幼苗,O2-含量低于对照植株,而3种酶的活性高于对照植株,均可有效地减轻SO₂的氧化胁迫.在SO₂熏蒸下,乙烯显著抑制3种酶的活力,提高O2-的形成速率.SA与乙烯同时使用时,SA几乎完全消除了乙烯的负面作用.     

Solvent effects on substrate and phosphate interactions with the (Na+ + K+)-ATPase

(Na+ + K+)-ATPase activity of a dog kidney enzyme preparation was markedly inhibited by 10-30% (v/v) dimethyl sulfoxide (Me2SO) and ethylene glycol (Et(OH)2); moreover, Me2SO produced a pattern of uncompetitive inhibition toward ATP. However, K+-nitrophenylphosphatase activity was stimulated by 10-20% Me2SO and Et(OH)2 but was inhibited by 30-50%. Me2SO decreased the Km for this substrate but had little effect on the Vmax below 30% (at which concentration Vmax was then reduced). Me2SO also reduced the Ki for Pi and acetyl phosphate as competitors toward nitrophenyl phosphate but increased the Ki for ATP, CTP and 2-O-methylfluorescein phosphate as competitors. Me2SO inhibited K+-acetylphosphatase activity, although it also reduced the Km for that substrate. Finally, Me2SO increased the rate of enzyme inactivation by fluoride and beryllium. These observations are interpreted in terms of the E1P to E2P transition of the reaction sequence being associated with an increased hydrophobicity of the active site, and of Me2SO mimicking such effects by decreasing water activity: (i) primarily to stabilize the covalent E2P intermediate, through differential solvation of reactants and products, and thereby inhibiting the (Na+ + K+)-ATPase reaction and acting as a dead-end inhibitor to produce the pattern of uncompetitive inhibition; inhibiting the K+-acetylphosphatase reaction that also passes through an E2P intermediate; but not inhibiting (at lower Me2SO concentrations) the K+-nitrophenylphosphatase reaction that does not pass through such an intermediate; and (ii) secondarily to favor partitioning of Pi and non-nucleotide phosphates into the hydrophobic active site, thereby decreasing the Km for nitrophenyl phosphate and acetyl phosphate, the Ki for Pi and acetyl phosphate in the K+-nitrophenylphosphatase reaction, accelerating inactivation by fluoride and beryllium acting as phosphate analogs, and, at higher concentrations, inhibiting the K+-nitrophenylphosphatase reaction by stabilizing the non-covalent E2.P intermediate of that reaction. In addition, Me2SO may decrease binding at the adenine pocket of the low-affinity substrate site, represented as an increased Ki for ATP, CTP and 3-O-methylfluorescein phosphate.     

The serine hydroxymethyltransferase of Plasmodium lophurae.

Plasmodium lophurae serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified and characterized by (NH4)2SO4 fractionation and chromatography on Sephadex G-100. The enzyme, precipitated by 3.0.3.3 M (NH4)2SO4, had a molecular weight of 68,300 as estimated by exclusion chromatography on G-100. The pH optimum of the enzyme was 6.8-7.6 in sodium phosphate-citrate buffer. Citrate stabilized the enzyme during storage in phosphate buffer at 4 C. The Km was 4.3 X 10(-3) M for L-serine and 2.5 X 10(-4) M for tetrahydrofolate.     

Unusual Behavior of Membrane Somatic Angiotensin-Converting Enzyme in a Reversed Micelle System

Properties of the membrane and soluble forms of somatic angiotensin-converting enzyme (ACE) were studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. The membrane enzyme with a hydrophobic peptide anchor was more sensitive to anions and to changes in pH and composition of the medium than the soluble enzyme without anchor. The activity of both forms of the enzyme in the reversed micelles significantly depended on the molarity of the buffer added to the medium (Mes-Tris-buffer, 50 mM NaCl). The maximum activity of the soluble ACE was recorded at buffer concentration of 20-50 mM, whereas the membrane enzyme was most active at 2-10 mM buffer. At buffer concentrations above 20 mM, the rate of hydrolysis of the substrate furylacryloyl-L-phenylalanyl-glycylglycine by both ACE forms was maximal at pH 7.5 both in the reversed micelles and in aqueous solutions. However, at lower concentrations of the buffer (2-10 mM), the membrane enzyme had activity optimum at pH 5.5. Therefore, it is suggested that two conformers of the membrane ACE with differing pH optima for activity and limiting values of catalytic constants should exist in the reversed micelle system with various medium compositions. The data suggest that the activity of the membrane-bound somatic ACE can be regulated by changes in the microenvironment.     

Carbonic anhydrase assay: strong inhibition of the leaf enzyme by CO2 in certain buffers

Apparent carbonic anhydrase activity in leaf extracts, measured as the rate of H+ production associated with the CO2 hydration reaction, varied by as much as 25-fold when the assay buffer was varied. Highest activities were usually recorded in barbitone buffer, with lower activities in imidazole, Tricine, Hepes, Tris, and phosphate buffers. The greatest differences were observed with the enzyme isolated from leaves of the monocotyledonous plants Zea mays (maize) and Triticum aestivum (wheat). Smaller differences were observed with carbonic anhydrase from dicotyledonous species and there was no effect on the erythrocyte enzyme. Leaf carbonic anhydrase activity measured by the mass spectrometric procedure was unaffected by varying the assay buffer. The low activity in certain buffers observed with the former assay system was found to be due to inhibition of the enzyme-catalyzed reaction by higher concentrations of CO2. Carbonic anhydrase from some sources was also strongly inhibited by certain inorganic and organic anions.     

Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase

Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H(2)O(2) exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris-HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35 min at 80 °C) and T(m) (from 73 to 77.5 °C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H(2)O(2) stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H(2)O(2) tolerance in concentrated buffer.     

一氧化氮供体硝普钠浸种缓解盐胁迫对小麦种子萌发的抑制作用

以小麦品种‘德抗961＇为材料,用NO供体硝普钠（SNP）浸种研究外源NO对盐胁迫下小麦种子萌发的影响.结果表明：0.06 mmol/L的SNP浸种24 h后对盐胁迫下小麦种子发芽率、发芽指数、活力指数和吸胀速率的下调都有显著缓解作用;SNP浸种对盐胁迫下α-淀粉酶的活性无明显影响,但能显著提高盐胁迫下β-淀粉酶的活性;进一步研究表明,SNP浸种预处理对盐胁迫下的α-淀粉酶同工酶变浅的条带有所恢复（尤其是条带3）,同时使盐胁迫下变浅的β-淀粉酶同工酶的条带有明显的恢复（尤其是d、e、f、g）.并且SNP能显著降低盐胁迫下小麦地上部分和根中的Na^＋含量,提高其K＋含量,从而使K^＋/Na^＋显著提高.以上结果表明：SNP浸种预处理提高盐胁迫下小麦种子的萌发,主要是通过提高β-淀粉酶的活性来实现的.     

Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa.

A proteinase from Pseudomonas aeruginosa exhibiting collagenolytic activity was purified 1575-fold with a recovery of 24% by use of chemical and chromatographic technics. The enzyme preparation appeared to be homogeneous when subjected to chromatographic, electrophoretic and ultracentrifugational analyses. A standard state sedimentation coefficient of 2.10 S was calculated and further analyses indicated that the enzyme had a molecular weight of 17 500 and dimerizes under certain conditions to yield an apparent molecular weight of 34 000. In addition to insoluble collagen, the enzyme catalyzed the hydrolysis of congocoll, azocoll, soluble collagen and casein, but did not attack orcein-elastin, azoalbumin, p-toluene eulfonyl-L-arginine methyl ester, benzoyl-L-tyrosine ethyl ester, and the hexapeptide N-benzyloxycarbonyl-glycyl-L-prolyglycylglycyl-L-prolyl-L-alanine. Enzymatic activity against congocoll was 6-fold greater at pH 7.5 in Tris with HCl than in phosphate buffer at the same ionic strength. Cobalt, and to a lesser extent, Zn2+ appeared to activate the enzyme, especially in phosphate buffer. NcCN and p-chloromercuribenzoate did not appreciably inhibit enzyme activity, while (NH4)2 SO4, EDTA and cysteine displayed a significant inhibitory effect under certain conditions.     

二氧化硫胁迫导致拟南芥防护基因表达改变

研究SO2熏气对拟南芥细胞中mRNA和蛋白质表达的影响,分析植株对逆境胁迫的响应机制.结果表明,30 mg·m-3 SO2 熏气72 h后拟南芥地上组织中差异表达1倍以上的基因有494个,其中抗氧化酶、谷胱甘肽硫转移酶(GST)、硫氧还蛋白等多种与逆境生理关系密切的基因表达上调;2.5～30 mg·m-3 SO2 熏气可导致超氧化物岐化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GPX)和GST的活性诱导性增高,SOD、CAT同工酶谱带特征改变.研究结果表明,SO2 胁迫能够诱导拟南芥中防护基因在mRNA和蛋白质表达水平的改变,这些基因的差异性表达可能对逆境生理过程有益.     

Joint action of l-leucine and sodium phosphate buffer solutions as feeding stimulants for protein-deprived females of the house fly Musca domestica

ABSTRACT. Potentiation in joint action was demonstrated between solutions of L-leucine and sodium phosphate buffer (pH 6.3) as feeding stimulants for protein-deprived females of the house fly, Musca domestica L. Both components alone elicited feeding. In two-choice feeding tests, mixtures consisting of equi-stimulating concentrations of the two components were taken in greater quantities than either component alone at twice the concentration in the mixture.
The presence of 1×10^-1 M phosphate buffer markedly lowered the threshold for detection of L-leucine. The presence of phosphate buffer strengthened the preferences shown by flies given choices of concentrations of L-leucine differing by a factor of 2 and enabled them to display preferences at lower concentrations.
The presence of 1×10^-3 M L-leucine increased, somewhat, the ability of flies to detect low concentrations of phosphate buffer. Its presence had relatively little effect on the strength of preference shown between two-fold differences in concentration of phosphate buffer when the higher concentration was 6.3×10^-3 M or less, but markedly strengthened the preferences when the higher concentration was 2.5×10^-2M or greater. Leucine increased the optimal concentration of phosphate buffer by a factor of more than 2 and converted 2×10^-1 M phosphate buffer from a mild feeding deterrent to a powerful feeding stimulant.     

微粒体细胞色素P-450、NADPH-细胞色素P-450还原酶的纯化与重组活性

经苯巴比妥钠诱导的雄性大白鼠的肝微粒体纯化的细胞色素P-450同功酶组份,经SDS-PAGE鉴定呈电泳纯,分子量为55kD。部分纯化的NADPH-细胞色素P-450还原酶,含72和77kD两个蛋白质组分。上述细胞色素P-450和NADPH-细胞色素P-450还原酶与卵磷脂制备的脂质体重组后的活性试验表明,对艾氏剂有环氧化作用,对环已烷有羟化作用,对溴氰菊酯的羟化作用微弱。当重组系统中缺少细胞色素P-450组份时,对环已烷不再起作用。同时还研究了纯化的细胞色素P-450的光谱特性。     

Fructose 1,6-diphosphate-activated L-lactate dehydrogenase from Streptococcus lactis: kinetic properties and factors affecting activation.

The L-(+)-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) of Streptococcus lactis C10, like that of other streptococci, was activated by fructose 1,6-diphosphate (FDP). The enzyme showed some activity in the absence of FDP, with a pH optimum of 8.2; FDP decreased the Km for both pyruvate and reduced nicotinamide adenine dinucleotide (NADH) and shifted the pH optimum to 6.9. Enzyme activity showed a hyperbolic response to both NADH and pyruvate in all the buffers tried except phosphate buffer, in which the response to increasing NADH was sigmoidal. The FDP concentration required for half-maximal velocity (FDP0.5V) was markedly influenced by the nature of the assay buffer used. Thus the FDP0.5V was 0.002 mM in 90 mM triethanolamine buffer, 0.2 mM in 90 mM tris(hydroxymethyl)aminomethanemaleate buffer, and 4.4 mM in 90 mM phosphate buffer. Phosphate inhibition of FDP binding is not a general property of streptococcal lactate dehydrogenase, since the FDP0.5V value for S. faecalis 8043 lactate dehydrogenase was not increased by phosphate. The S. faecalis and S. lactis lactate dehydrogenases also differed in that Mn2+ enhanced FDP binding in S. faecalis but had no effect on the S. lactis dehydrogenase. The FDP concentration (12 to 15 mM) found in S. lactis cells during logarithmic growth on a high-carbohydrate (3% lactose) medium would be adequate to give almost complete activation of the lactate dehydrogenase even if the high FDP0.5V value found in 90 mM phosphate were similar to the FDP requirement in vivo.     

The investigation of substrate-induced changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenases by measurement of the kinetics and thermodynamics of subunit exchange.

1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.     

Studies on the Pectolytic Enzyme

Pectinesterase was extracted from the pulp of tomato fruit (Lycopersicum esculentum var. Hikari) pericarp with 250 mM potassium phosphate buffer, pH 8.0, and purified about 60 folds by means of ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100 column. The enzyme preparation thus obtained was confirmed to be homogeneous state both ultracentrifugationally and disk electrophoretically. The sedimentation coefficient of this enzyme was calculated to be 3.17 S.     

甲醇对酵母过氧化氢酶活性的影响机理研究

将酵母过氧化氢酶加入一定比例的甲醇,测定其活性变化。结果表明:在含2%甲醇时酶活比对照提高4026%。将粗酶液用70%饱和度的硫酸铵盐析后离心所得的上清液再加入硫酸铵至80%饱和度,离心的沉淀溶解在缓冲液中,上SephadexG75柱,分离出的有酶活性的蛋白峰经电泳得一条蛋白带,说明过氧化氢酶已经被提纯到电泳纯。光谱分析发现,甲醇处理后过氧化氢酶纯酶的吸收光谱和荧光发射光谱与未经处理的比较基本不变,而差示光谱出现明显的正峰和负峰。由动力学分析可知,在甲醇中,过氧化氢酶的Vmax和Km值均有不同程度提高 。     

Increase of electrical properties using a novel mixed buffer system in an enzyme fuel cell

Environment-friendly biocatalytic energy is considered to represent an attractive alternative to chemical catalystbased cells due to its renewability and better operation at low temperature. However, electrical biocatalysts have a low activity and electrical power. For increasing electrical properties of biocatalyst, a novel mixed buffer (phosphate and 3-morpholinopropanesulfonic acid (MOPS)) system was applied to an enzyme-based biofuel cell with microperoxidase (MP-11)-modified Au electrode. The cathodic electrical properties were increased by the phosphate and MOPS-mixed buffer solution. It was identified that the novel mixed buffer system obtained stronger ionic strength from phosphate buffer and better enzyme activity from MOPS buffer. The highest results of cyclic voltammetry were obtained when the proportion of phosphate to MOPS was nearly 1:1 and the pH was 7.0∼7.3. In addition, the novel mixed buffer led to the maximum power density (ca. 62.7 μW/cm²) in a basic enzymatic fuel cell (EFC).     

镉诱导的菊芋幼苗膜脂过氧化和抗氧化酶活性及过氧化物酶同工酶的改变

用含有不同浓度(0~400μmol/L)Cd(NO3)2的Hoagland营养液处理砂培的菊芋。处理50d后,测定植物体内镉积累量以及过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性,并对POD同工酶进行电泳分析。发现在Cd50~100μmol/L浓度内,随着镉浓度的升高,菊芋根和叶中镉的积累量显著增加,而随后积累量的增加有所减少。根和叶中MDA含量显著上升,说明镉引起了膜脂过氧化。0~100μmol/LCd处理,根和叶中POD活性随Cd浓度增加而增强,而在200~400μmol/LCd处理下有所减弱。根和叶SOD活性在50~200μmol/LCd处理下随Cd浓度增加而增强,而在400μmol/LCd处理下SOD活性明显受到抑制。根和叶CAT活性随Cd浓度升高而增强。电泳结果显示,POD同工酶变化明显,镉诱导出一条新酶带LP10。菊芋POD同工酶可以作为镉污染的土壤的生物指示剂。     

磷酸缓冲液对植物脂质过氧化的影响及其与亚硫酸伤害的关系

以亚硫酸模式处理不同类型植物 ,从脂质过氧化方面探讨了磷酸缓冲液的防护作用 .结果表明 ,植物经磷酸缓冲液处理后 ,对亚硫酸伤害具有明显的防护作用 .磷酸缓冲液可稳定细胞膜结构 ,降低MDA含量 ,增强叶绿素———蛋白质的结合度 ,且与低浓度亚硫酸对SOD活性呈协同效应 .磷酸缓冲液的防护作用因植物种类而异 .同一污染环境下 ,MDA含量高 ,叶绿素结合度低者效应显著 .在测试植物中 ,银杏 >连翘 >小麦苗 .