Biotransformation of D‐panthenol and its action in protection of activation of oxidizing processes in brain

It is a well known fact that 3H‐panthenol (PL) has a high bioavailability, so we studied its biotransformation and its protective action against lipoperoxide activation in homogenates and mitochondrial‐synaptosomal fraction (11 000 g) of rat brain. The lipoperoxidation was initialized by Fe²⁺‐ascorbate complex (Fe²⁺‐Asc). In experiments in vivo, after 30 min, we demonstrated accumulation of intermediate products of CoA biosynthesis – pantothenic acid (PA), phospho‐PA, and phosphopantetheine – in postmitochondrial fraction of brain, by using a HPLC technique. Addition of the PL (10 mm ) to brain hemispheres homogenates or mitochondrial‐synaptosomal fraction caused a remarkable reduction of malondialdehyde production. However, 30 min preincubation with the PL, but not with PA, was ineffective. The data obtained may be a reason for a high neuroprotective activity of PL in curing brain diseases with vessel or alcohol‐induced damages.     

Phytase activity of Selenomonas ruminantium: a preliminary characterization

Obligately anaerobic ruminal bacteria have been found to possess phytase activity, in particular, Selenomonas ruminantium . The phytase activity of S. ruminantium JY35 was produced late in growth and required neither phytate for induction nor phosphate limitation for derepression. The activity was completely cell-associated with a significant fraction extractable by a magnesium chloride solution. Zymogram analysis suggested that the activity was the result of a single gene product of a monomeric nature and approximately 46 kDa in size. The phytase had a temperature optimum of 50–55 °C, but activity dropped off sharply at 60 °C. Phytase activity was optimal over the pH range of 4·0–5·5, and dependent on the nature of the buffer used. Activity was inhibited by citric acid buffer and by the addition of 5 mmol l⁻¹ Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺ and Hg²⁺. The addition of 5 mmol l^–1 Pb²⁺ to the enzyme assay appeared to enhance activity of the enzyme.     

Plasma membrane-bound H+-ATPase and reductase activities in Fe-deficient cucumber roots

Physiological and biochemical modifications induced by Fe-deficiency have been studied in cucumber ( Cucumis sativus L. cv. Marketer) roots, a Strategy I plant that initiates a rapid acidification of the medium and an increase in the electric potential difference when grown under Fe-deficiency. Using the aqueous two-phase partitioning method, a membrane fraction which has the plasmalemma characteristics was purified from roots of plants grown in the absence and in the presence of iron. The plasma membrane vesicles prepared from Fe-deficient plants showed an H⁺-ATPase activity (EC 3.6.1.35) that is twice that of the non-deficient control. Furthermore, membranes from Fe-deficient plants showed a higher capacity to reduce Fe³⁺-chelates. The difference observed in the reductase activity was small with ferricyanide (only 30%) but was much greater with Fe³-EDTA and Fe³-citrate (210 and 250%, respectively). NADH was the preferred electron donor for the reduction of Fe³⁺ compounds. Fe³⁺ reduction in plasma membrane from cucumber roots seems to occur with utilisation of superoxide anion, since addition of superoxide dismutase (SOD; EC 1.15.1.1) "in vitro" decreased Fe³⁺ reduction by 60%.
The response and the difference induced by iron starvation on these two plasma membrane activities together with a possible involvement of O₂ in controlling the Fe³⁺/Fe²⁺ ratio in the rhizosphere are discussed.     

Indispensability of Iron for the Growth of Cultured Chick Cells

In order to clarify the role of iron in the growth promoting effect of transferrin (Tf), the effects of the following substances were examined in cultured chick skeletal myogenic cells: transition metal ions (Fe²⁺, Fe³⁺, Cr³⁺, Cu²⁺, Mn²⁺, Co²⁺, Cd²⁺, Zn²⁺ and Ni²⁺), Tf complexes with these metals and metal-free apoTf.
The cells did not grow well when incubated in a culture medium composed of Eagle's minimum essential medium and horse serum. But they grew well in the presence of Fe²⁺ or Fe³⁺ (10–100 μM) or iron-bound Tf (10–500 nM) in the medium. None of the transition metal ions other than iron was effective. Neither apoTf nor Tf complexes with these metals showed the growth promoting effect. The generality of the requirement of iron for cell growth was ascertained in the primary culture of other types of chick embryonic cells: fibroblasts, cardiac myocytes, retinal pigment cells and spinal nerve cells.
The results show that iron is one of the indispensable substances for cell growth and suggest that Tf protein plays a role in facilitating the transport of iron into the cells.     

Electrical signalling and cytokinins mediate effects of light and root cutting on ion uptake in intact plants

Nutrient acquisition in the mature root zone is under systemic control by the shoot and the root tip. In maize, exposure of the shoot to light induces short-term (within 1–2 min) effects on net K⁺ and H⁺ transport at the root surface. H⁺ efflux decreased (from −18 to −12 nmol m⁻² s⁻¹) and K⁺ uptake (∼2 nmol m⁻² s⁻¹) reverted to efflux (∼−3 nmol m⁻² s⁻¹). Xylem probing revealed that the trans-root (electrical) potential drop between xylem vessels and an external electrode responded within seconds to a stepwise increase in light intensity; xylem pressure started to decrease after a ∼3 min delay, favouring electrical as opposed to hydraulic signalling. Cutting of maize and barley roots at the base reduced H⁺ efflux and stopped K⁺ influx in low-salt medium; xylem pressure rapidly increased to atmospheric levels. With 100 m m NaCl added to the bath, the pressure jump upon cutting was more dramatic, but fluxes remained unaffected, providing further evidence against hydraulic regulation of ion uptake. Following excision of the apical part of barley roots, influx changed to large efflux (−50 nmol m⁻² s⁻¹). Kinetin (2–4  µ m ), a synthetic cytokinin, reversed this effect. Regulation of ion transport by root-tip-synthesized cytokinins is discussed.     

Characterization of soluble rifamycin oxidase from Curvularia lunata var. aeria

Curvularia lunata var. aeria was grown on yeast extract, peptone and carboxymethylcellulose (YPC) medium for the production of extracellular rifamycin oxidase. The enzyme was partially purified through a Sephadex G-75 column. The half lives of rifamycin oxidase at 30° and 40°C were 9 d and 100 min, respectively. The activation and deactivation energies of the partially purified enzyme, calculated from Arrhenius plots, were 5.80 and 35.10 kcal mol^-1 respectively. The enzyme exhibited a K _m (rifamycin B) value of 0.67 mmol l^-1 and a V _max of 11 μmol h^-1 ml. Three metal ions, Fe²⁺, Ag⁺ and Hg²⁺, inhibited the enzyme in the 10–20 mmol l^-1 metal ion concentration range. Catalytic activity was not affected by the chelating agent, EDTA.     

PYRUVATE METABOLISM BY HOMOGENATES OF HUMAN BRAIN: EFFECTS OF PHENYLPYRUVATE AND IMPLICATIONS FOR THE ETIOLOGY OF THE MENTAL RETARDATION IN PHENYLKETONURIA

Abstract— The effect of phenylalanine and phenylpyruvate on the metabolism of pyruvate by homogenates of human brain was investigated. In the presence of 5 mM pyruvate as substrate homogenates of human cerebral cortex fixed about 1 μmol of H¹⁴CO₃^-_- per g of tissue in 30 min. Phenylpyruvate at a concentration of 5 raw inhibited the fixation of H¹⁴ CO₃^-_- by homogenates of human brain by approximately 50 per cent, whereas 5 mM phenylalanine had no effect. The inhibition of pyruvate carboxylation by phenylpyruvate was dependent upon the concentration of the inhibitor. The activity of pyruvate carboxylase (EC 6.4.1.1) in human cerebral cortex was 02–0.4 units, with a K_m for pyruvate of about 0.2 mM. Homogenates of human cerebral cortex decarboxylated [1-¹⁴C]pyruvate to ¹⁴CO₂ at a rate of about 5 μmol per g of tissue per 15 min, with a 20–50 per cent reduction in the presence of 5 mM phenylpyruvate; phenylalanine at the same concentration had no effect. The possible toxic effect of phenylpyruvate on the metabolism of pyruvate in the brains of untreated phenylketonuric patients is discussed.     

Purification and characterization of the major β-1,4-endoglucanase from Thermomonospora curvata

The major β-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata , contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose. The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC. This monomeric enzyme had a specific activity of 750 IU mg⁻¹ when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70°C, pH 6.0. Highest activity was observed on CMC with a degree of polymerization of 3200. The EG was stable for 48 h at 60°C, pH 6.0 and had a half-life of 30 min at 80°C; temperature and pH optima were 70–73°C and 6.0–6.5, respectively. The mol. wt was 100000 and the pI was 4.0. The K _m and V _max values were 7.33 mg ml⁻¹ and 833 μmol min⁻¹, respectively. EG activity was inhibited by Fe^{2 +}, Hg^{2 +}, Ag⁺ and Pb^{2 +}, and enhanced by dithiothreitol and Zn^{2 +}. The first 12 amino acid residues at the N -terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser. Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found.     

The Effect of Potassium Nitrate on the Reduction of Phytophthora Stem Rot Disease of Soybeans, the Growth Rate and Zoospore Release of Phytophthora sojae

The effects of potassium nitrate (KNO₃) application on Phytophthora stem rot disease reduction of Glycine max (L.) Merr. cvs. Chusei-Hikarikuro and Sachiyutaka, and mycelium growth and zoospore release of a Phytophthora sojae isolate were investigated under laboratory conditions. The application of 4–30 m m KNO₃ prior to inoculation greatly reduced incidence of disease in the two soybean cultivars. Although a concentration of 20–30 m m KNO₃ led to a slight decrease in the growth rate of the PJ-H30 isolate on PDA medium, no significant relationship was observed between inhibition of the growth rate and disease reduction on application of 0.4–10 m m KNO₃. Disease suppression recorded in laboratory experiments using pathogen mycelium was due to the response of plant tissues rather than a direct inhibition of pathogen hyphal growth by the application of KNO₃. The extent of disease reduction was related to increased potassium concentration in plants of the two cultivars (except for some cases involving cv. Sachiyutaka), suggesting that differences existed between the two cultivars in terms of the effect of KNO₃ application on disease suppression. Scanning electron microscopic observation with fresh samples indicated marked accumulation of potassium at the penetration-stopping sites of P. sojae in the cortex layer of soybean plants treated with 30 m m KNO₃, compared with the non-treated control plants. The presence of 0.4–30 m m KNO₃ decreased the release of zoospores. These results suggest the possibility of applying a solution containing 20–30 m m of KNO₃ to decrease the incidence of disease in agricultural fields by the response of plant tissues to KNO₃.     

Effects of saponin extracts from Solanum elaeagnifotium fruits on ion uptake by clover seedlings

Solanum elaeagnifolium Cav. fruits contain high concentrations of steroidal saponins. Treatment of 3-day-old clover seedlings with aqueous fruit extracts modified Ca²⁺ uptake without significantly altering K⁺ and H₂PO₄⁻ uptake. The extracts increased Ca²⁺ uptake in the concentration range of 0.2 to 20 m M Ca²⁺. Uptake curves could be represented by two phases. In the lower phase (0.2-1.0 m M Ca²⁺), this change could be related to an increase in V_max. Pretreatment of seedlings with saponin extracts significantly reduced ATP-dependent Ca²⁺ uptake and Ca²⁺-dependent ATPase activity in a fraction isolated from root homogenates by centrifugation at 1500 g for 15 min. Saponins purified from S. eleagnifolium extracts by thin-layer chromatography modified in vitro the Ca²⁺-ATPase activity of this fraction, indicating that the steroid may act directly on Ca²⁺ transport across membranes.     

New insights into the metal specificity of the Pseudomonas aeruginosa pyoverdine–iron uptake pathway

Pyoverdine (PvdI) is the major siderophore secreted by Pseudomonas aeruginosa PAOI in order to get access to iron. After being loaded with iron in the extracellular medium, PvdI is transported across the bacterial outer membrane by the transporter, FpvAI. We used the spectral properties of PvdI to show that in addition to Fe³⁺, this siderophore also chelates, but with lower efficiencies, all the 16 metals used in our screening. Afterwards, FpvAI at the cell surface binds Ag⁺, Al³⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe³⁺, Ga³⁺, Hg²⁺, Mn²⁺, Ni²⁺ or Zn²⁺ in complex with PvdI. We used Inductively Coupled Plasma-Atomic Emission Spectrometry to monitor metal uptake in P. aeruginosa : TonB-dependent uptake, in the presence of PvdI, was only efficient for Fe³⁺. Cu²⁺, Ga³⁺, Mn²⁺ and Ni²⁺ were also transported into the cell but with lower uptake rates. The presence of Al³⁺, Cu²⁺, Ga³⁺, Mn²⁺, Ni²⁺ and Zn²⁺ in the extracellular medium induced PvdI production in P. aeruginosa . All these data allow a better understanding of the behaviour of the PvdI uptake pathway in the presence of metals other than iron: FpvAI at the cell surface has broad metal specificity at the binding stage and it is highly selective for Fe³⁺ only during the uptake process.     

Uptake of Cd2+ and Fe2+ by excised roots of Tamarix aphylla

The uptake of Cd²⁺ by excised roots of Tamarix aphylla (L.) Karst, was investigated using roots of hydroponically grown plants. The concentration isotherm of Cd²⁺ uptake approached saturation with a single phase hyperbola. The time course of Cd²⁺ absorption was generally hyperbolic, with an apparent linear section between 2 and 30 min. The temperature response varied among different temperature ranges: a Q₁₀ of approximately 1.9 was found between 10 and 20°C, but at higher and lower temperatures Q₁₀ values were only 1–1.3. It is concluded that Cd²⁺ uptake by the roots of T. aphylla at moderate temperatures is mediated by a metabolic process, combined with a passive influx component that becomes dominant at higher and lower temperatures. The distribution of the absorption sites for Cd²⁺ and for Fe²⁺ along the roots of T. aphylla was also investigated. Cadmium uptake showed no apparent pattern, whereas a distinct pattern of uptake was observed for Fe²⁺, with the highest rates at the root tip. Iron absorption was stimulated in the presence of nutrients, whereas that of Cd²⁺ was inhibited. Adsorption and absorption of Cd²⁺ were strongly inhibited by Ca²⁺ and by Mg²⁺, but were unaffected by Fe²⁺. Monovalent ions (Na⁺, K⁺, Li⁺) also reduced Cd²⁺ absorption, but to a lesser extent than Ca²⁺ and Mg²⁺. Uptake of Cd⁺ was reduced at lower pH of the medium. The importance of interfering cations for Cd²⁺ tolerance of T. aphylla is emphasized.     

Degeneration of biogenic superparamagnetic magnetite

Magnetite crystals precipitated as a consequence of Fe(III) reduction by Shewanella algae BrY after 265 h incubation and 5-year anaerobic storage were investigated with transmission electron microscopy, Mössbauer spectroscopy and X-ray diffraction. The magnetite crystals were typically superparamagnetic with an approximate size of 13 nm. The lattice constants of the 265 h and 5-year crystals are 8.4164Å and 8.3774Å, respectively. The Mössbauer spectra indicated that the 265 h magnetite had excess Fe(II) in its crystal-chemistry (Fe³⁺_1.990Fe²⁺_1.015O₄) but the 5-year magnetite was Fe(II)-deficient in stoichiometry (Fe³⁺_2.388Fe²⁺_0.419O₄). Such crystal-chemical changes may be indicative of the degeneration of superparamagnetic magnetite through the aqueous oxidization of Fe(II) anaerobically, and the concomitant oxidation of the organic phases (fatty acid methyl esters) that were present during the initial formation of the magnetite. The observation of a corona structure on the aged magnetite corroborates the anaerobic oxidation of Fe(II) on the outer layers of magnetite crystals. These results suggest that there may be a possible link between the enzymatic activity of the bacteria and the stability of Fe(II)-excess magnetite, which may help explain why stable nano-magnetite grains are seldom preserved in natural environments.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

The mntH gene encodes the major Mn2+ transporter in Bradyrhizobium japonicum and is regulated by manganese via the Fur protein

The bacterial Nramp family protein MntH is a divalent metal transporter, but mntH mutants have little or no phenotype in organisms where it has been studied. Here, we identify the mntH homologue of Bradyrhizobium japonicum , and demonstrate that it is essential for Mn²⁺ transport and for maintenance of cellular manganese homeostasis. Transport activity was induced under manganese deficiency, and Fe²⁺ did not compete with ⁵⁴Mn²⁺ for uptake by cells. The steady-state level of mntH mRNA was negatively regulated by manganese, but was unaffected by iron. Control of mntH expression and Mn²⁺ transport by manganese was lost in a fur strain, resulting in constitutively high activity. Fur protected a 35 bp region of the mntH promoter in DNase I footprinting analysis that includes three imperfect direct repeat hexamers that are needed for full occupancy. Mn²⁺ increased the affinity of Fur for the mntH promoter by over 50-fold, with a K _d value of 2.2 nM in the presence of metal. The findings identify MntH as the major Mn²⁺ transporter in B. japonicum , and show that Fur is a manganese-responsive regulator in that organism. Furthermore, Fe²⁺ is neither a substrate for MntH nor a regulator of mntH expression in vivo .     

Guinea pig hippocampal 5-HT1E receptors: a tool for selective drug development

Recent studies have indicated that the serotonin [5-hydroxytryptamine (5-HT)] 1E receptor, originally discovered in human brain tissue, is not expressed in rat or mouse brain. Thus, there have been few reports on 5-HT_1E receptor drug development. However, expression of 5-HT_1E receptor mRNA has been shown in guinea pig brain. To establish this species as an animal model for 5-HT_1E drug development, we identified brain regions that exhibit 5-carboxyamidotryptamine, ritanserin, and LY344864 – insensitive [³H]5-HT binding (characteristic of the 5-HT_1E receptor). In hippocampal homogenates, where 5-HT_1E receptor density was sufficiently high for radioligand binding analysis, 100 nM 5-carboxyamidotryptamine, 30 nM ritanserin, and 100 nM LY344864 were used to mask [³H]5-HT binding at non-5-HT_1E receptors. The K _d of [³H]5-HT was 5.7 ± 0.7 nM and is indistinguishable from the cloned receptor K _d of 6.5 ± 0.6 nM. The affinities of 16 drugs for the cloned and hippocampal-expressed guinea pig 5-HT_1E receptors are essentially identical ( R ² = 0.97). These findings indicate that using these conditions autoradiographical distribution and signal transduction studies of the 5-HT_1E receptor in guinea pig brain are feasible. Using the guinea pig as an animal model should provide important insights into possible functions of this receptor and the therapeutic potential of selective human 5-HT_1E drugs.     

SOME CHARACTERISTICS OF ATPase ACTIVITY IN A BRAIN MICROTUBULE PROTEIN PREPARATION

Abstract— The Mg- and Ca-ATPase activities in a brain tubulin preparation have been measured. The activity of the microtubule protein (MTP) preparation was optimal, 3-4.5 nmol Pi/mg protein/min, at pH 8.0 in the presence of 1-2 m m -Mg²⁺ or Ca²⁺, with a half maximal stimulation at about 0.3 m m concentration of either of the divalent ions.
Phosphocellulose (PC) purified tubulin exhibited no or very low activity (0-2 nmol Pi/mg protein/min).
The majority of ATPase activity was found in the microtubule associated proteins (MAP) fraction. It was stimulated by Mg²⁺ and Ca²⁺, inhibited by NaF or high ionic strength but unaffected by vanadate at 10⁻⁴ m . In decreasing order of effectiveness ATP, GTP, UTP, CTP and ADP were hydrolyzed. p -Npp was a poor substrate. V_max values for Mg- and Ca-ATPase activities were about 15 and 10 nmol Pi/mg protein/min, respectively with a K_m value of about 25 μ m . However, double reciprocal plots disclosed more complicated kinetics, which were not fully resolved.
The activity was largely confined to 30-36S material (i.e.'rings'and 'spirals'). The protein responsible for the ATPase activity is possibly the smaller one of the two (or three) high molecular weight (HMW) proteins of mol wt over 200,000.
There are similarities between this enzyme and both flagellar dynein and myosin. However, the present ATPase differs from myosin in several important aspects (i.e. ionic requirements). Furthermore, no peptides of the myosin type were found upon electrophoretic analysis of the MAP fraction.     

Resting and arecoline-stimulated brain metabolism and signaling involving arachidonic acid are altered in the cyclooxygenase-2 knockout mouse

Studies were performed to determine if cyclooxygenase (COX)-2 regulates muscarinic receptor-initiated signaling involving brain phospholipase A₂ (PLA₂) activation and arachidonic acid (AA; 20 : 4n-6) release. AA incorporation coefficients, k* (brain [1–¹⁴C]AA radioactivity/integrated plasma radioactivity), representing this signaling, were measured following the intravenous injection of [1–¹⁴C]AA using quantitative autoradiography, in each of 81 brain regions in unanesthetized COX-2 knockout (COX-2^–/–) and wild-type (COX-2^+/+) mice. Mice were administered arecoline (30 mg/kg i.p.), a non-specific muscarinic receptor agonist, or saline i.p. (baseline control). At baseline, COX-2^–/– compared with COX-2^+/+ mice had widespread and significant elevations of k*. Arecoline increased k* significantly in COX-2^+/+ mice compared with saline controls in 72 of 81 brain regions, but had no significant effect on k* in any region in COX-2^–/– mice. These findings, when related to net incorporation rates of AA from brain into plasma, demonstrate enhanced baseline brain metabolic loss of AA in COX-2^–/– compared with COX-2^+/+ mice, and an absence of a normal k* response to muscarinic receptor activation. This response likely reflects selective COX-2-mediated conversion of PLA₂-released AA to prostanoids.     

Metabolism of Prostaglandin D2 by Human Cerebral Cortex into 9α, 11β-Prostaglandin F2 by an Active NADPH-Dependent 11-Ketoreductase

Abstract: In homogenates of rat cerebral neocortex prostaglandin D₂ (PGD₂) was found to be quantitatively the main PG biosynthesized by a cytosolic PGD synthetase from en-dogenously released arachidonic acid. Amounts of 628 ng/g wet weight were found after 30-min incubation periods compared with basal levels of 2.3 ng/g wet weight. In human cerebral cortex, whether obtained at biopsy or postmortem, only small amounts of PGD₂ (4.5–11.7 ng/g wet weight/30 min) were formed. Furthermore, PGD₂, added to homogenates of human biopsy temporal cortex, was converted efficiently into 9α,11β-PGF₂ by a NADPH-dependent 11-ke-toreductase as has been reported in other human tissues (liver and lung). PGF_2α was determined directly as the fl-butylbo-ronate derivative. It became clear that 9α,11β-PGF₂ was formed in considerably greater amounts than PGF_2α and that other metabolites are also formed. These results can account for the low amounts of PGD₂ found in incubations of human brain tissue. The rat brain does not contain 11-ketoreductase activity. The present results indicate that the 9α, 11β-PGF₂ must be considered along with other eicosanoids in pathophysiological situations in brain.     

ATP-dependent H+-pumping of a low-density fraction of pea stem microsomes

A low-density fraction of pea ( Pisum sativum L. cv. Alaska) stem microsomes, obtained from a discontinuous sucrose gradient, possessed an H⁺-ATPase able to generate a proton gradient and an electrical potential. The proton pumping was insensitive to monovalent cations, to vanadate and oligomycin, required a permeant anion and was inhibited by nitrate, N, N'-dicyclohexylcarbodiimide and diethylstilbestrol. The H⁺-ATPase had a pH optimum around 6.0–6.5 and was saturable with respect to the substrate Tris-ATP (K_m≅ 0.4 m M ). Ca²⁺ (0.05–1 m M ) induced a dissipation of the ATP-generated δpH without affecting ATPase activity. At physiological concentrations (1–5 m M ), nitrate caused an initial slight increase of the ATP-generated proton gradient followed by a complete dissipation after 2–3 min. The dissipating effect was not caused by inhibition of ATPase activity, since ATP prevented the nitrate-induced collapse of δpH. On the other hand, ATPase activity, evaluated as release of P_i, was not inhibited by concentrations lower than 20 m M KNO₃. These results indicate that nitrate entered the vesicles in response to an electrical potential and then could exit in symport with protons, while Ca²⁺ entered in exchange for protons (antiport).