Selective Inhibition of Auxin-Stimulated NADH Oxidase Activity and Elongation Growth of Soybean Hypocotyls by Thiol Reagents

The NADH oxidase activity of isolated vesicles of soybean (Glycine max cv Williams 82) plasma membranes and elongation growth of 1-cm-long hypocotyl segments were stimulated by auxins (indole-3-acetic acid or 2,4-dichlorophenoxyacetic acid [2,4-D]). The auxin-induced stimulations of both NADH oxidase and growth were prevented by the thiol reagents N-ethylmaleimide, p-chloromercuribenzoate, 5,5[prime]-dithiobis(2-nitrophenylbenzoic acid), dithiothreitol, and reduced glutathione. These same reagents largely were without effect on or stimulated slightly the basal levels of NADH oxidase and growth when assayed in the absence of auxins. In the presence of dithiothreitol or reduced glutathione, both 2,4-D and indole-3-acetic acid either failed to stimulate or inhibited the NADH oxidase activity. The rapidity of the response at a given concentration of thiol reagent and the degree of inhibition of the 2,4-D-induced NADH oxidase activity were dependent on order of reagent addition. If the thiol reagents were added first, auxin stimulations were prevented. If auxins were added first, the inhibitions by the thiol reagents were delayed or higher concentrations of thiol reagents were required to achieve inhibition. The results demonstrate a fundamental difference between the auxin-stimulated and the constitutive NADH oxidase activities of soybean plasma membranes that suggest an involvement of active-site thiols in the auxin-stimulated but not in the constitutive activity.     

Activation of Plasma Membrane NADH Oxidase Activity by Products of Phospholipase A

An auxin-stimulated NADH oxidase activity (NADH oxidase I) of plasma membrane vesicles, highly purified by aqueous two-phase partition from soybean (Glycine max Merr.) hypocotyls was activated by lysophospholipids and fatty acids, both products of phospholipase A action. The activation of NADH oxidase activity occurred slowly, suggesting a mechanism whereby the lipids acted to stabilize the enzyme in a more active configuration. In contrast to activation by lipids, the activation by auxin was rapid. The average K_m of the NADH oxidase after activation by lipids was four- to fivefold less than the K_m before activation. The V_max was unchanged by activation. The increases occurred in the presence of detergent and thus were not a result of exposure of latent active sites. Also, the activation did not result from activation of a peroxidase or lipoxygenase. Fatty acid esters, where growth promoting effects have been reported, also activated the auxin-stimulated oxidase. However, the auxin stimulation of NADH oxidase I did not appear to be obligatorily mediated by phospholipase A, nor did inhibitors of phospholipase A₂ block the stimulation of the oxidase by auxins.     

NADH Oxidase Activity of Plasma Membranes of Soybean Hypocotyls Is Activated by Guanine Nucleotides

The activity of an auxin-stimulated NADH oxidase of the plasma membrane of hypocotyls of etiolated soybean (Glycine max Merr.) seedlings responded to guanine and other nucleotides, but in a manner that differed from that of enzymes coupled to the classic trimeric and low molecular weight monomeric guanine nucleotide-binding proteins (G proteins). In the presence and absence of either auxin or divalent ions, both GTP and GDP as well as guanosine-5[prime]-O-(3-thiotriphosphate) (GTP-[gamma]-S) and other nucleoside di- and triphosphates stimulated the oxidase activity over the range 10 [mu]M to 1 mM. GTP and GTP-[gamma]-S stimulated the activity at 10 nM in the absence of added magnesium and at 1 nM in the presence of added magnesium ions. Other nucleotides stimulated at 100 nM and above. The NADH oxidase was stimulated by 10 [mu]M mastoparan and by 40 [mu]M aluminum fluoride. Neither cholera nor pertussis toxins, tested at a concentration sufficient to block mammalian G protein function, inhibited the activity. Guanosine 5[prime]-O-(2-thiodi-phosphate) (GDP-[beta]-S) did not stimulate activity, suggesting that the stimulation in response to GDP may be mediated by a plasma membrane nucleoside diphosphate kinase through conversion of GDP to GTP. Auxin stimulation of the NADH oxidase was unaffected by nucleotides at either high or low nucleotide concentrations in the absence of added divalent ions. However, pretreatment of plasma membranes with auxin increased the apparent affinity for nucleotide binding. This increased affinity, however, appeared not to be the mechanism of auxin stimulation of the oxidase, since auxin stimulation was similar with or without low concentrations of guanine nucleotides. The stimulation by nucleotides was observed after incubating the membranes with 0.1% Triton X-100 prior to assay. The results suggest a role of guanine (and other) nucleotides in the regulation of plasma membrane NADH oxidase that differs from the interactions with G proteins commonly described for animal models.     

b-Type Dihydroorotate Dehydrogenase Is Purified as a H2O2-Forming NADH Oxidase from Bifidobacterium bifidum

Our previous report showed the existence of microaerophilic Bifidobacterium species that can grow well under aerobic conditions rather than anoxic conditions in a liquid shaking culture. The difference in the aerobic growth properties between the O₂-sensitive and microaerophilic species is due to the existence of a system to produce H₂O₂ in the growth medium. In this study, we purified and characterized the NADH oxidase that is considered to be a key enzyme in the production of H₂O₂. Bifidobacterium bifidum, an O₂-sensitive bacterium and the type species of the genus Bifidobacterium, possessed one dominant active fraction of NADH oxidase and a minor active fraction of NAD(P)H oxidase activity detected in the first step of column chromatography for purification of the enzyme. The dominant active fraction was further purified and determined from its N-terminal sequence to be a homologue of b-type dihydroorotate dehydrogenase (DHOD), composed of PyrK (31 kDa) and PyrDb (34 kDa) subunits. The genes that encode PyrK and PryDb are tandemly located within an operon structure. The purified enzyme was found to be a heterotetramer showing the typical spectrum of a flavoprotein, and flavin mononucleotide and flavin adenine dinucleotide were identified as cofactors. The purified enzyme was characterized as the enzyme that catalyzes the DHOD reaction and also catalyzes a H₂O₂-forming NADH oxidase reaction in the presence of O₂. The kinetic parameters suggested that the enzyme could be involved in H₂O₂ production in highly aerated environments.     

Interaction of Partially Purified Phytotoxins from Phytophthora cactorum on Apple Cell Plasma Membrane

The ‘crude’ filtrate (CF) of Phytophthora cactorum containing phytotoxin(s), having some properties similar to the toxins isolated from other Phytophthora species, was processed by three steps (acetone precipitation, dialysis and gel filtration chromatography). The CF fractions corresponding to the progressive steps of purification were tested for phytotoxicity on tomato seedlings and for activity on cell trans-membrane electrical potential (Em) of susceptible and resistant apple rootstocks (Malus domestica). The fractions (F4), obtained from chromatography on Sephadex G50 fine and eluted in the zone corresponding to a molecular weight of 15 ± 2 kD, induced a specific alteration on susceptible apple cell membranes. These metabolites, even though incompletely purified, are able to induce a high and specific activity on susceptible apple cell Em only, not on resistant ones. As a consequence, they may be of potential use in screening for insensitive cells.     

Touch-Sensitive NADH Oxidase Activity of Pea and Cucumber Tendrils and Soybean Hypocotyl Sections

The cell surface reduced nicotinamide adenine dinucleotide (NADH) oxidase activity of soybean stems and of pea and cucumber tendrils responded to touch with a several-fold increase in activity. The increase in NADH oxidase persisted for 20 min or longer, and further touch stimulation during this period did not alter activity. With soybean sections, the specific activities in response to touch approximated those achieved maximally by auxin. Where the NADH oxidase was fully stimulated by 2,4-d, the NADH oxidase failed to respond further to touch. The findings indicate that the NADH oxidase of the plant cell surface is involved in the growth response to touch and in tendril coiling.     

烟草—TMV相互作用中质膜NADPH氧化酶的组装

比较 Ｎ Ｎ 烟草（与烟草花叶病毒（ Ｔ Ｍ Ｖ）发生非亲和相互作用）和普通烟草 ３００２ 品种（与 Ｔ Ｍ Ｖ 发生亲和相互作用）在烟草— Ｔ Ｍ Ｖ 的相互作用中质膜 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶的组装激活、产生活性氧的差异．用两相法制备密闭的正向型质膜（ Ｐ Ｍ）囊泡,以 Ｓ Ｏ Ｄ 敏感的 Ｎ Ａ Ｄ Ｐ Ｈ 依赖的 Ｃｙｔ ｃ 的还原表示 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶的活性,用人类噬中性白细胞 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶亚基 ｐ４７ ｐｈｏｘ 的抗体对烟草叶片蛋白进行免疫学检测．结果显示在两种烟草叶片胞质中均存在与 ｐ４７ ｐｈｏｘ 亚基的抗体发生免疫交叉反应的相同分子量的蛋白,该蛋白在 Ｔ Ｍ Ｖ 侵染 Ｎ Ｎ 基因烟草后可向质膜发生转移,且伴随有氧化酶活性的升高．而对于普通烟草则无氧化酶膜组分和酶活性的明显变化．以上结果表明,烟草叶片质膜上存在与哺乳动物 Ｎ Ａ Ｄ Ｐ Ｈ 氧化酶相类似的氧化酶,它的组装和激活可能是烟草— Ｔ Ｍ Ｖ 非亲和相互作用早期活性氧的主要来源．     

双水相纯化的人肺腺癌和癌旁正常肺组织质膜蛋白质组差异分析

肿瘤标志物对于肺腺癌病人的临床诊断和预后具有重要意义. 本研究根据临床诊断选取人肺腺癌组织和癌旁正常肺组织为研究对象,采用差速离心联合双水相法纯化组织细胞质膜,运用同位素标记相对和绝对定量技术结合高效液相色谱 串联质谱技术,鉴定出肺腺癌组织和癌旁正常肺组织的差异蛋白质41种. 同癌旁正常肺组织相比,18个蛋白质在肺腺癌组织中表达上调,23个蛋白质在肺腺癌组织中表达下调. 生物信息学分析发现,差异质膜蛋白质FLOT1、CAV1和ITGB1均处于蛋白质相互作用网络的重要位置,可能参与了肺腺癌相关信号转导.利用蛋白质印迹和免疫组织化学染色验证,差异蛋白质FLOT1、CAV1和ITGB1在肺腺癌织和癌旁正常肺组织的表达情况,其验证结果与蛋白质组学研究结果一致. 研究结果对肺腺癌诊断标志物和肺腺癌癌变分子机理研究具有重要意义.     

Plasma Membrane NADPH Oxidase in Tobacco tobacco mosaic virus Interaction

The plasma membrane NADPH oxidase and its regulatory role in the production of reactive oxygen species (ROS) in tobacco (Nicotiana tabacum L. )-tobacco mosaic virus (TMV) interaction was examined by using tobacco cv. "Samsun NN" (incompatible with TMV, containing the N gene for resistance to TMV) and tobacco cv. "3002" (compatible with TMV) as experimental materials. Plasma membrane (PM) vesicles were isolated from leaves of tobacco by a biphasic aqueous system. The membrane preparations were sealed, highly purified and largely in right-side-out orientation as detected by marker enzyme assays and latency studies of the PM marker, vanadate-sensitive ATPase with non-ionic detergent Triton X-100. The oxidase activity was assayed by the rate of SOD-sensitive Cyt c reduction in PM system. The oxidase activity could be increased about 80% when adding 0.01% Triton X-100 in the reactive system. This result showed that the binding-site of NADPH was on the cytosolic side of the plasma membrane and the production of O2- is on the apoplastic side. DPI (diphenylene iedonium), a specific inhibitor of the NADPH oxidase in neutrophils, also inhibited the NADPH oxidase activity in tobacco. Furthermore, the oxidase activity increased in incompatible interaction, but not in compatible interaction. The role of NADPH oxidase in the production of reactive oxygen species and stimulation of hypersensitive reaction were discussed.     

大豆下胚轴质膜H+-ATPase质子转运的测定

以大豆下胚轴为材料,采用改进的匀浆介质,通过两相法制得具有质子转运活力的高纯度质膜微囊.并且发现冻融处理可以促进质膜微囊的翻转而提高荧光猝灭效率.质子载体和质子转运特性分析表明,由Mg^2＋-ATP引发的荧光猝灭可以被质子载体CCCP恢复,并被质子通道抑制剂DCCD抑制;并且发现质膜H^＋-ATPase专一抑制剂钒酸钠可以完全抑制荧光猝灭,同时发现荧光猝灭依赖于Mg^2＋,并受K^＋刺激,最适pH为6.5.以上证明所测荧光猝灭是由质膜H^＋-ATPase所进行的质子转运引起的.结果同时表明,维持H^＋-ATPase合适构象和提高质膜微囊封闭性是制备具有H^＋转运活力质膜微囊的两个关键因素.     

Diacylglycerol Levels Unchanged during Auxin-Stimulated Growth of Excised Hypocotyl Segments of Soybean

Diacylglycerol contents of excised soybean (Glycine max L.) hypocotyl segments, incubated for 4 hours in the presence or absence of a growth promoting concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) were monitored by three different methods as a sensitive measure of the action in vivo of C-type phospholipases. By all three methods, steady state levels of diacylglycerols representing about 3% of the total lipids or about 7% of the neutral lipids, depending on method of assay, declined 18% over 4 hours of incubation as determined by extraction of total lipids and analysis by thin layer chromatography and densitometry. The average decline with 2,4-D-treated segments was less but the difference from controls was not significant. In those experiments where a small effect of 2,4-D was noted, the fraction showing an elevated diacylglycerol level in response to 2,4-D, after separation into membrane and supernatant fractions, was the supernatant and not the membranes. Results were confirmed from analyses of total fatty acids in each of the major lipid fractions and from diacylglycerol assays by conversion into phosphatidic acid upon incubation with [γ-³²P]ATP and purified diacylglycerol phosphokinase from Escherichia coli. In the presence of 2,4-D, the diacylglycerol content of the membranes was unchanged compared to membranes from control segments. As with the densitometric method, the small 2,4-D induced increase in diacylglycerols, when observed, was insignificant and in the supernatant. The only membrane-associated lipid fraction consistently showing a response to 2,4-D was the fraction containing sterols esterified with fatty acids. Either total microsomes or purified plasma membranes when incubated for 10 to 20 minutes with 1 micromolar 2,4-D showed no accelerated formation of diacylglycerols compared to membranes not incubated. The results do not support operation during auxin growth of the animal paradigm where diacylglycerol activation of C-type protein kinases occurs in response to activated phospholipase C breakdown of phosphoinositides.     

大豆叶片质膜蛋白激酶的自身磷酸化反应

利用Ferrell和Martin（1991）设计的测定印迹在PVDF膜上的蛋白激酶活性方法研究大豆叶片质膜蛋白激酶自身磷酸化反应活性,结果表明：与Mg－ATP相比,Mn－ATP是更有效的57KD蛋白激酶自身磷酸化反应底物;钙离子可以促进该激酶的自身磷酸化反应活性,而且EGTA可以显著降低它在SDS电泳中的迁移率,说明57KD蛋白激酶为依赖于钙的蛋白激酶;预磷酸化反应实验证明57KD蛋白激酶具有多个自身磷酸化反应位点,其分子的自身磷酸化状态可调性暗示这一激酶可能具有重要的生理功能。     

Characteristics and Regulatory Properties of the H+-ATPase in a Plasma Membrane Fraction Purified from Arabidopsis thaliana

The aqueous two-phase partitioning technique was utilized to isolate a plasma membrane (PM) fraction from etiolated seedlings of Arabidopsis thaliana. The purification procedure adopted yielded a fraction highly enriched in PM as compared to inner membranes, with a recovery of about 30%, as judged from the activities of PM markers such as vanadate-sensitive ATPase, FC binding and UDP-glucose sterol glucosyltransferase. The purified PM fraction displayed vanadate-sensitive H⁺ pumping activity. Its purity was confirmed by the biochemical characteristics of its ATPase activity assayed in the absence of Ca²⁺: sensitivity to vanadate (IC₅₀ ca. 1 μM), Mg²⁺-dependence, insensitivity to molybdate, oligomycin and nitrate, pH optimum at 6.6. The PM H⁺-ATPase activity was stimulated by fusicoccin and by a controlled treatment of the PM with trypsin. In both cases stimulation was much stronger on the activity assayed at pH 7.5 than on the activity at pH 6.6. Moreover, neither fusicoccin nor the treatment with trypsin stimulated the portion of activity (30 to 40% at pH 7.5) which decayed upon preincubation of the PM in assay medium without ATP.     

Fusicoccin Binding to its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase. II. Stimulation of the H+-ATPase in a Plasma Membrane Fraction Purified by Phase-partitioning

The effect of fusicoccin (FC) on the activity of the PM H⁺-ATPase was investigated in a plasma membrane (PM) fraction from radish seedlings purified by the phase-partitioning procedure. FC stimulated the PM H⁺-ATPase activity by up to 100 %; the effect was essentially on V_max with only a slight decrease of the apparent K_M of the enzyme for ATP. FC-induced stimulation of the PM H⁺-ATPase was evident within the first minute and maximal within five minutes of membrane treatment with the toxin indicating that transmission of the signal from the activated receptor to the PM H⁺-ATPase is very rapid. Both FC-induced stimulation of the PM H⁺-ATPase and FC binding to its receptor decreased dramatically upon incubation of the membranes in ATPase assay medium at 33 °C in the absence of FC, due to the lability of the free FC receptor. FC-induced stimulation of the PM H⁺-ATPase was strongly pH dependent: absolute increase of activity was maximal at pH 7, while percent stimulation increased with the increase of pH up to pH 7.5; FC binding was scarcely influenced by pH in the pH range investigated. Taken as a whole, these results indicate that FC binding is a condition necessary, but not sufficient, for FC-induced stimulation of the PM H⁺-ATPase.     

Location of the Electron Accepting Site of a b-Type Cytochrome Oxidase in Purified Chromatophore Membranes and Spheroplast Membrane Vesicles from Photosynthetically Grown Rhodopseudomonas sphaeroides

Purified chromatophore membranes and spheroplast membrane vesicleswere prepared from Rhodopseudomonas sphaeroides by the methodof Michels and Konings (1978). The cytochrome c oxidase activityof the spheroplast membrane vesicles was about 15-fold higheron a bacteriochlorophyll basis than that of the chromatophoremembranes. The cytochrome c oxidase activity of the chromatophoremembranes was greatly stimulated (about 10-fold) by an additionof Triton X-100 (0.1%), but there was less stimulated (about1.5-fold) in the case of spheroplasts. The pH optimum of theoxidase activities of the spheroplast membrane vesicles andof chromatophore membranes treated with 0.1% Triton X-100, andthe salt dependencies of the activity of both preparations werethe same. These results show that the membrane-bound b-type cytochromeoxidase of this bacterium accepts electrons from ferrocytochromec on the periplasmic side of the membrane (on the outer surfaceof the spheroplast membrane vesicles). These results also areconsistent with the fact that cytochrome c₂ is located in theperiplasmic space in this bacterium. (Received January 24, 1984; Accepted April 24, 1984)     

Blue Light Exposure Targets NADPH Oxidase to Plasma Membrane and Nucleus in Wheat Coleoptiles

Wheat coleoptile tips generate superoxide radical as a part of the phototropic response to blue light, but the source of this free radical generation is not known. We evaluated the presence and involvement of homologs of neutrophil NADPH oxidase (NOX), including gp91phox, p22phox, p67phox, p47phox, and p40phox, in wheat coleoptiles using Western blot analysis and immunofluorescence microscopy. Blue light augmented the expression levels of all these subunits and targeted NOX subunits onto the plasma membrane and to the nucleus. gp91phox, p22phox, p67phox, and p40phox showed entry into the nucleus and exhibited physical closeness with DNA. CuZnSOD was also present in the coleoptile tip, which also showed a blue-light-dependent elevation in expression. Superoxide production and phototropic response were both abrogated by DPIC and staurosporine, indicating their cause-and-effect relationship. We conclude that blue light mediates a phototropic response in wheat coleoptiles through modulation of expression of NOX and SOD as well as the translocation of NOX subunits onto the plasma membrane and nuclear membrane. Thus, this study provides a mechanistic explanation for superoxide production during the photoresponse in wheat coleoptiles.     

Sterol Modulation of the Plasma Membrane H+-ATPase Activity from Corn Roots Reconstituted into Soybean Lipids

A partially purified H+-ATPase from the plasma membrane (PM) of corn (Zea mays L.) roots was inserted into vesicles prepared with soybean (Glycine max L.) phospholipids and various concentrations of individual sterols using either a freeze-thaw sonication or an octylglucoside dilution procedure. Both methods yielded a functional enzyme that retained its native characteristics. We have investigated the effects of typical plant sterols (i.e. sitosterol, stigmasterol, and 24-methylcholesterol) on both ATP hydrolysis and H+ pumping by the reconstituted corn root PM ATPase. We have also checked the influence of cholesterol and of two unusual sterols, 24-methylpollinastanol and 14[alpha],24-dimethylcholest-8-en-3[beta]-ol. Here we present evidence for a sterol modulation of the plant PM H+-ATPase activity. In particular, cholesterol and stigmasterol were found to stimulate the pump, especially when present at 5 mol%, whereas all of the other sterols tested behaved as inhibitors at any concentration in proteoliposomes. In all situations H+ pumping was shown to be more sensitive to a sterol environment than was ATP hydrolysis. Our results suggest the occurrence of binding sites for sterols on the plant PM H+-ATPase.