Cytochemical localization of glutaraldehyde-resistant NAD(P)H-oxidase in rat hepatocytes

NAD(P)H-oxidase activity was demonstrated in glutaraldehyde-fixed rat hepatocytes by a cerium technique. The activity was observed exclusively on the bile-canalicular plasma membrane of hepatocyte. No reaction product was formed in the absence of NAD(P)H as the substrate. The reaction was inhibited by pCMB (surface sulfhydryl group specific reagent), by heating, by anaerobic incubation and by catalase (H2O2 scavenger), but it was not inhibited by KCN or NaN3. The present results show that bile-canalicular plasma membrane produces H2O2 and the cerium technique for demonstration of H2O2 is therefore an useful method for the subcellular localization of NAD(P)H-oxidase activity in the glutaraldehyde-fixed hepatocyte.     

APPEARANCE AND FUNCTION OF ENDOGENOUS PEROXIDASE IN FETAL RAT THYROID

Iodination within the thyroid follicle is intimately associated with a thyroid peroxidase. In order to locate the in vivo site of iodination, the initial cytochemical appearance of this enzyme has been determined in fetal rat thyroid and its presence correlated with the onset of iodinated thyroglobulin synthesis. Peroxidase first appears in follicular cells during the 18th day of gestation. It is seen first in the perinuclear cisternae, the cisternae of the endoplasmic reticulum, and within the inner few Golgi lamellae. These organelles presumably represent sites of peroxidase synthesis. During the 19th and 20th days of gestation, there is a tremendous increase in peroxidase activity. In addition to the stained sites described, there are now many peroxidase-positive apical vesicles in the follicular cells. Newly forming follicles stain most conspicuously for peroxidase, the reaction product being heavily concentrated at the external surfaces of apical microvilli and in the adjacent colloid. Iodinated thyroglobulin becomes biochemically detectable in thyroids during the 19th day of gestation and increases greatly during the 20th day. The parallel rise in peroxidase staining that just precedes, and overlaps, the rise in iodinated thyroglobulin, suggests that apical vesicles and the apical cell membrane are the major sites of iodination within the thyroid follicle.     

Cytochemical localization of plasma membrane enzyme markers during interiorization of tachyzoites of Toxoplasma gondii by macrophages

The enzyme activity of Mg++-ATPase, Na+-K+-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected at the ultrastructural level in mouse peritoneal macrophages infected with untreated and with specific antibody-coated Toxoplasma gondii tachyzoites. The Mg++-ATPase and 5'-nucleotidase were distributed throughout the macrophages' plasma membrane but were not observed in the membrane lining endocytic vacuoles containing ingested parasites; however, Na+-K+-ATPase activity was detected in the macrophages' plasma membrane as well as in the parasitophorous vacuoles that contained untreated or specific antibody-coated parasites. Reaction product, indicative of NAD(P)H-oxidase, was detected in the parasitophorous vacuoles that contained only specific antibody-coated parasites.     

NAD(P)H-oxidase presence in Toxoplasma gondii tachyzoite vacuole during interaction with IFN-gamma-activated human endothelial cells

Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells (HUVEC) where it resides in a parasitophorous vacuole (PV) preventing lysosomal fusion. To study the intracellular outcome of PV containing tachyzoites of T. gondii during interaction with IFN-gamma-activated HUVEC, a quantitative analysis of the T. gondii infection and multiplication was assayed. The quantification of PVs' fusion with lysosomes, ultrastructural examination of phagosome-lysosome fusion, and the localization of NAD(P)H-oxidase activity were also investigated. HUVEC activated with IFN-gamma inhibited T. gondii infection and multiplication by 67.5% and 91.0%, respectively. After 4 hr of infection, 10.2% of IFN-gamma-activated HUVEC exhibited phagosome-lysosome fusion assayed by fluorescence microscopy, which was also observed at the ultrastructural level. Furthermore, the enzyme NAD(P)H-oxidase present at the plasma membrane of activated HUVEC was internalized together with the parasite in 38.0% of the cells. In addition, colocalization of colloidal gold particles and reaction product of NAD(P)H-oxidase in the PV of some activated HUVEC was observed. These results suggest that NAD(P)H-oxidase may participate in a mechanism by which IFN-gamma-activated HUVEC inhibit T. gondii multiplication.     

Cytochemical localization of hydrogen peroxide production in the rat uterus

A reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent H2O2-generating activity of the rat uterus was investigated both electron cytochemically and biochemically. We tried to cytochemically demonstrate H2O2 generation from the oxidation of reduced NADH or NADPH using the cerium method. NADPH oxidation resulted in electron-dense deposits on the apical plasma membrane covering the microvilli of the surface epithelium of the lightly fixed endometrium. In control specimens incubated in a medium from which substrate was omitted, no such deposits were observed. The reduction of ferricytochrome c due to NADH oxidation was spectrophotometrically detected in the lightly fixed uterus. Absorption at 550 nm increased with the addition of NADH, but not with that of NAD. The reaction was weakened by preheating and adversely affected by the addition of superoxide dismutase, but it was not inhibited by adding 50 mM sodium azide. These results suggest that a kind of NAD(P)H oxidase, generating H2O2 via superoxide formation, may possibly be present on the apical plasma membrane of the rat endometrial epithelium.     

Partial purification of a plasma membrane flavoprotein and NAD(P)H-oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H-dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two-phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg^?1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non-covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H-oxidoreductase activity and suggests the presence of plasma membrane bound pterins.     

质膜NAD(P)H氧化酶参予金瓜炭疽(Colletotrichum lagerarium)细胞壁激发子诱导人参细胞产生激发反应(英文)

在人参(Panax ginseng C.A.Meyer)悬浮细胞质膜上测出了NAD(P)H氧化酶活性。这类NAD(P)H氧化酶活性可以被金瓜炭疽细胞壁激发子(Cle)诱导。Cle处理还能诱导人参悬浮细胞的氧进发、促进人参悬浮细胞的皂苷合成、提高苯丙氨酸解氨酶(PAL)的活力、以及诱导查尔式酮酶(CHS)的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp(Hydroxyprolin-rich glycoproleins)的表达。当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二亚苯基碘(Diphenylene iodonium,DPI)与奎吖因(quinacrine)预处理人参悬浮细胞30 min 后,Cle诱导的H2O2释放与Cle激活的质膜NAD(P)H氧化酶活性被抑制,同时Cle诱导的PAL活性及CHS的积累下降,皂苷合成与hrgp的表达被抑制。由此推测:人参细胞质膜NAD(P)H氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性。在Cle激发人参悬浮细胞产生氧进发的过程中,NAD(P)H氧化酶活性被诱导从而导致H2O2的产生,H2O2作为第二信使,激活苯丙氨酸途径,诱发人参皂苷的合成及hrgp防御基因的表达。这一过程中还涉及到Ca2+内流,胞内Ca2+浓度的升高,蛋白磷酸化与去磷酸化。人参细胞质膜NAD(P)H氧化酶在人参细胞对Cle的反应过程中起一种介导作用。因此可能存在由Cle刺激,NAD(P)H氧化酶被诱导,H2O2释放,到人     

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells cultivated aerobically or anaerobically

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells were examined on the basis of the cell growth rate, NAD(P)H concentration, reactive oxygen production, plasma membrane H⁺-ATPase activity, and ethanol production. In aerobically or anaerobically cultured yeast cells, NAD(P)H concentration decreased with increasing concentration of menadione, and the recovery of NAD(P)H concentration was proportional to the cell growth rate. However, there was no relationship among the inhibition of the cell growth and reactive oxygen production, plasma membrane H⁺-ATPase activity, and ethanol production. Among them, ethanol production showed resistance to the cytotoxicity of menadione, suggesting the resistance of glycolysis to menadione. The growth inhibitory effect of menadione depended on the rapid decrease and the recovery of NAD(P)H rather than production of reactive oxygen species regardless of aerobic culture or anaerobic culture and presence or absence of mitochondrial function. The recovery of NAD(P)H concentration after the addition of menadione might depend on menadione-resistant glycolytic enzymes.     

Cytochemical Localization of Plasma Membrane Enzyme Markers During Interiorization of Tachyzoites of Toxoplasma gondii by Macrophages

The enzyme activity of Mg-ATPase, Na⁺-K⁺-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected at the ultrastructural level in mouse peritoneal macrophages infected with untreated and with specific antibody-coated Toxoplasma gondii tachyzoites. The Mg⁺⁺-ATPase and 5'-nucleotidase were distributed throughout the macrophages'plasma membrane but were not observed in the membrane lining endocytic vacuoles containing ingested parasites; however, Na⁺-K⁺-ATPase activity was detected in the macrophages'plasma membrane as well as in the parasitophorous vacuoles that contained untreated or specific antibody-coated parasites. Reaction product, indicative of NAD(P)H-oxidase, was detected in the parasitophorous vacuoles that contained only-specific antibody-coated parasites.     

Possible interaction between peroxidase and NAD(P)H-dependent nitrate reductase activities of plasma membranes of corn roots

The relationship between the plasma membrane bound NAD(P)H-nitratereductase (NR) and a plasma membrane (PM)-bound peroxidase wasinvestigated using highly purified PM vesicles isolated fromcorn roots. The PM-bound NR activity was strongly enhanced byMnCl₂ and SHAM, which stimulated peroxidase activity. Sinceboth activities, the NAD(P)H-dependent NR and the peroxidasecompete for NAD(P)H as electron donor, we propose a model inwhich a product of peroxidation is able to offer electrons tothe nitrate reductase in a more reactive form with respect toNAD(P)H.Our hypothesis was confirmed by experiments in which the effectsof inhibitors of peroxidative reactions, catalase, superoxidedismutase, and ascorbate on the PM-bound NR were studied. Resultsindicate that the putative electron donor for nitrate reductioncould be a radicalic species, possibly NAD. Furthermore, sincecytochrome c decreased the activity of the plasma membrane-boundNAD(P)Hdependent NR, cytochrome b₅₅₇ might be the site of theenzyme accepting electrons from NAD. Our results indicate that the PM environment of the NR may beinvolved in the extent of the membrane associated nitrate reductionand that redox enzymes at the PM, the NAD(P)H-NR and a peroxidase-likeNADH-oxidase, can interact. Key words: Plasma membrane-bound nitrate reductase, peroxidase, Zea mays     

Myeloperoxidase-catalyzed iodination and coupling.

Myeloperoxidase (MPO), which displays considerable amino acid sequence homology with thyroid peroxidase (TPO) and lactoperoxidase (LPO), was tested for its ability to catalyze iodination of thyroglobulin and coupling of two diiodotyrosyl residues within thyroglobulin to form thyroxine. After 1 min of incubation in a system containing goiter thyroglobulin, I-, and H2O2, the pH optimum of MPO-catalyzed iodination was markedly acidic (approximately 4.0), compared to LPO (approximately 5.4) and TPO (approximately 6.6). The presence of 0.1 N Cl- or Br- shifted the pH optimum for MPO to about 5.4 but had little or no effect on TPO- or LPO-catalyzed iodination. At pH 5.4, 0.1 N Cl- and 0.1 N Br- had a marked stimulatory effect on MPO-catalyzed iodination. At pH 4.0, however, iodinating activity of MPO was almost completely inhibited by 0.1 N Cl- or Br-. Inhibition of chlorinating activity of MPO by Cl- at pH 4.0 has been previously described. When iodination of goiter thyroglobulin was performed with MPO plus the H2O2 generating system, glucose-glucose oxidase, at pH 7.0, the iodinating activity was markedly increased by 0.1 N Cl-. Under these conditions iodination and thyroxine formation were comparable to values observed with TPO. MPO and TPO were also compared for coupling activity in a system that measures coupling of diiodotyrosyl residues in thyroglobulin in the absence of iodination. MPO displayed very significant coupling activity, and, like TPO, this activity was stimulated by a low concentration of free diiodotyrosine (1 microM). The thioureylene drugs, propylthiouracil and methimazole, inhibited MPO-catalyzed iodination both reversibly and irreversibly, in a manner similar to that previously described for TPO-catalyzed iodination.     

NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Peroxidase and tyrosinase are present in secondary lysosomes that degrade photosensory membranes of the crayfish photoreceptor: possible role in pigment granule formation.

Three enzymes (acid phosphatase, peroxidase, and tyrosinase) were localized by electron microscopy within the retina of crayfish Orconectes limosus. Peroxidase activity was observed only in lamellar bodies, which are secondary lysosomes and degrade photosensory membrane. After H2O2 was omitted from the reaction medium, peroxidase activity in lamellar bodies was partly inhibited but was not missing completely. After addition of sodium pyruvate, which inhibits endogenous generation of H2O2, staining of lamellar bodies was absent. Tyrosinase activity was found in lamellar bodies and in small vesicles within the rhabdoms similar to those found positive for acid phosphatase. Granules (500-700 nm in diameter) with an electron opaque matrix and mature screening pigment granules showed tyrosinase activity. Moreover, lamellar structures within membrane-bound organelles that additionally contained screening pigment-like granules were electron dense because of tyrosinase activity. After addition of phenylthiourea (PTU) to the incubation medium, lamellar bodies did not generally contain electron dense deposits, although weak staining of single membranes still was sometimes observed. After addition of sodium pyruvate in combination with PTU, no staining was detected. The possible role of tyrosinase in ommochrome synthesis within secondary lysosomes that degrade photosensory membrane is discussed.     

NAD(P)H-utilizing oxidoreductases of the plasma membrane An overview of presently purified proteins

Summary A considerable number of studies have demonstrated the presence of NAD(P)-oxidoreductases in the plant and animal cell plasma membranes. Recently several attempts on the isolation and purification of these proteins have been presented. The results indicate the presence of distinct NAD(P)H-utilizing enzymes in the plasma membrane of several species. Proteins with molecular masses of 27 kDa, 31 kDa, 36–39 kDa, and 45 kDa have been identified. Little information is so far available on the presence and nature of the chromophores on these proteins. The electron donor and acceptor specificities of the purified enzymes seem to depend to some extent on the purification procedures used. Two interesting remarks became apparent when evaluating the literature available on this subject. First, although some plasma membrane NAD(P)H-oxidoreductase activity is transmembrane, none of the purified enzymes was reported to depend on the presence of polar lipids to reach full activity. Second, considerable amounts of enzyme activity were found in the non-solubilised membrane material and apparently resisted the solubilisation procedures. The nature of these activities has not yet been clarified. Clearly the amino acid sequencing and structural analysis of these proteins will reveal important new clues to the understanding of the plasma membrane electron transport in the near future.Abbreviations DQ duroquinone - HCF hexacyanoferrate (III)     

Hormonogenesis in thyroid cells cultured on porous bottom chambers

Different processes implied in thyroid hormonogenesis (thyroglobulin, thyroperoxidase and hydrogen peroxide generating system expressions) and their regulation by TSH and iodide have been studied using porcine thyroid cells cultured in porous bottomed chambers. This system allowed to reproduce the functional bipolarity. Cells form a tight and polarized monolayer. Both apical and basolateral poles of epithelial cells were independently accessible and the cell layer separated two compartments which can contain different media. A major polarized secretion of thyroglobulin into the apical compartment was observed; it was increased in the presence of TSH as well as the thyroglobulin synthesis and mRNA level. These TSH effects were the consequence of adenylcyclase stimulation. Active transport of iodide, iodination of thyroglobulin and hormonosynthesis took place only in the presence of TSH. These steps occurred at the apical pole of cells. In the culture chamber system, thyroglobulin was weakly iodinated (6 atoms of iodide per mole of thyroglobulin; in vivo up to 40 atoms per mole) but hormonogenesis efficiency was close to this one observed in vivo (40%). Iodide concentrations higher than 0.5 µM daily added to the basal medium inhibited iodination of thyroglobulin and hormonosynthesis. Some components contained in culture media were inhibitors for iodination when they were present in the apical medium such as vitamins, amino acids and phenol red. The culture system appears to be interesting for pharmacological and toxicological studies.     

Process of iodination of thyroglobulin and its maturation. I. Properties and distribution of thyroglobulin labeled with radioiodine in pig thyroid slices.

Pig thyroid slices were incubated with Na131I and the 17--19S 131I-labeled thyroglobulin isolated was subjected to dissociation with 0.3 mM sodium dodecyl sulphate SDS) on sucrose density gradient centrifugation and to iodoamino acid analysis. During the incubation, initially dissociable thyroglobulin was gradually altered to 0.3 mM SDS-resistant species with increasing incorporation of iodine. Microsome-bound, poorly iodinated thyroglobulin and preformed thyroglobulin were chemically iodinated and then subjected to analysis of dissociability and iodoamino acid contents with newly incorporated iodine. The results indicated that the behavior of the former thyroglobulin resembled that of 131I-thyroglobulin obtained from the slices. Then, thyroid slices were incubated for 3 min with Na131I and 3H-leucine with or without 10-min chase incubation. The sucrose density gradient centrifugation patterns of 131I and 3H-radioactivity of cytoplasmic extracts indicated that 131I-thyroglobulin is contained in particulates, especially in vesicles with low density(d=1.12) and that some of them are released into the soluble fraction within 10 min. The vesicles contained peroxidase and NADH-cytochrome c reductase, and are probably exocytotic vesicles in the apical area of cytoplasm of follicular cells. No positive evidence was obtained that plasma membranes participate in the iodination of thyroglobulin under the present experimental conditions. These results suggest that, in the incubation of thyroid slices, iodine atoms are preferentially incorporated into newly synthesized, less iodinated thyroglobulin, rather than preformed thyroglobulin, and that the iodination occurs, at least to a certain degree, in apical vesicles before the thyroglobulin is secreted into the colloid lumen.     

Transcytosis in thyroid follicle cells

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.     

Effect of defoliant (butiphose) on morpho-physiological properties and enzyme systems of natural membranes

Butiphose (Tributyltritiophosphate, (C4H9S)3PO) a commonly used defoliant in cotton growing regions of USSR, caused extensive alterations in morphological features of erythrocyte and nuclear membranes and affected the permeability properties of rat liver mitochondrial membrane. It disrupted Ca2+ transport system and other energy dependent processes in mitochondria. A reduction in the activity of cytochrome-c-oxidase and NAD.H-oxidase was also observed.     

Immunocytochemical localization of protein kinases and calmodulin in dog thyroid cells

By immunocytochemical methods, the protein effectors of known intracellular signal molecules were demonstrated in the thyroid follicular cell and their localization investigated. Cyclic AMP protein kinase subunit RII was clearly located in the nucleus. Protein kinase subunits RI and C were in the cytosol and on the apical membrane. Cyclic GMP kinase and calmodulin were mostly found in the cytoplasm and at the apical membrane; they were poorly represented in the nucleus. The only membrane underlined by several markers was the apical membrane, i.e. the site of iodination, oxido reduction and H2O2 generation.     

Zinc deficiency enhanced NAD(P)H-dependent superoxider radical production in plasma membrane vesicles isolated from roots of bean plants

The production of superoxide radical (O₂^–) was studiedin plasma membrane vesicles isolated by aqueous polymer two-phasepartitioning from roots of zinc-sufficient and zinc-deficientbean (Phaseolus vulgaris L. cv. Prélude) plants. Thetwo populations of vesicles were highly enriched in plasma membraneand had similar composition as evidenced by the specific membranemarker enzymes. Vesicles from zinc-deficient roots showed higherrates of NAD(P)H oxidation compared to vesicles from zinc-sufficientplants. The NAD(P)H-dependent formation of O₂^– in plasmamembrane vesicles was also highly increased by zinc deficiency.For both activities, a higher response to zinc deficiency wasobserved when NADPH was used as electron source. Re-supply ofzinc to deficient plants for 24 h substantially decreased therates of NAD(P)H oxidation and 0₂^– production in isolatedvesicles. The NADPH-dependent O₂^– generation was stronglystimulated by FAD and showed a high pH optimum; it was scarcelyaffected by Triton X-100 or even inhibited in the presence ofFAD and was almost insensitive to Antimycin A. The results suggest the presence at the plasma membrane of beanroots of an O₂^– generating activity, preferentially utilizingNADPH, which is affected by the zinc nutritional status of theplant. This finding, together with previous observations oncytosolic and microsomal fractions prepared from zinc-deficientroots of different plants, is consistent with a role of zincin membrane stabilization by controlling the level of oxidizingO₂ species. Key words: NAD(P)H oxidase, superoxide radical, plasma membrane, zinc deficiency