Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.     

Competing peroxidase and oxidase reactions in scopoletin-dependent H2O2-initiated oxidation of NADH by horseradish peroxidase

Addition of NADH inhibited the peroxidative loss of scopoletin in presence of horseradish and H₂O₂ and decreased the ratio of scopoletin (consumed):H₂O₂ (added). Concomitantly NADH was oxidized and oxygen was consumed with a stoichiometry of NADH:O₂ of 2:1. On step-wise addition of a small concentration of H₂O₂ a high rate of NADH oxidation was obtained for a progressively decreasing time period followed by termination of the reaction with NADH:H₂O₂ ratio decreasing from about 40 to 10. The rate of NADH oxidation increased linearly with increase in scopoletin concentration. Other phenolic compounds including p-coumarate also supported this reaction to a variable degree. A 418-nm absorbing compound accumulated during oxidation of NADH. The effectiveness of a small concentration of H₂O₂ in supporting NADH oxidation increased in presence of SOD and decreased in presence of cytochrome c, but the reaction terminated even in their presence. The results indicate that the peroxidase is not continuously generating H₂O₂ during scopoletin-mediated NADH oxidation and that both peroxidase and oxidase reactions occur simultaneously competing for an active form of the enzyme.     

The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH

NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca²⁺ and pH in vitro, but concentrations of Ca²⁺ needed are not known. We have determined the K_0.5(Ca²⁺) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O₂ and decylubiquinone as electron acceptors. The K_0.5(Ca²⁺) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K_0.5(Ca²⁺) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K_0.5(Ca²⁺) values were determined at pH 6.8. Lower K_0.5(Ca²⁺) values were observed with decylubiquinone than with O₂ as terminal electron acceptor. NADPH oxidation responded to changes in Ca²⁺ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca²⁺-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.     

Formation and Conversion of Oxygen Metabolites by Lactococcus lactis subsp. lactis ATCC 19435 under Different Growth Conditions

A semidefined medium based on Casamino Acids allowed Lactococcus lactis ATCC 19435 to grow in the presence of oxygen at a slow rate (0.015 h⁻¹). Accumulation of H₂O₂ in the culture prevented a higher growth rate. Addition of asparagine to the medium increased the growth rate, whereby H₂O₂ accumulated only temporarily during the lag phase. H₂O₂ is an inhibitor for several glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase being the most sensitive. Strain ATCC 19435 contained NADH oxidase (maximum specific rate under aerobic conditions, 426 nmol of NADH min⁻¹ mg of protein⁻¹), which reduced oxygen to water, whereby superoxide was formed as a by-product. H₂O₂ originated from the dismutation of superoxide by superoxide dismutase. Although H₂O₂ was rapidly destroyed under high metabolic fluxes, neither NADH peroxidase nor any other enzymatic H₂O₂-reducing activity was detected. However, pyruvate, the end product of glycolysis, reacted nonenzymatically and rapidly with H₂O₂ and hence was a potential alternative for scavenging of this oxygen metabolite intracellularly. Indeed, intracellular concentrations of up to 93 mM pyruvate were detected in aerobic cultures growing at high rates. It is hypothesized that self-generated pyruvate may serve to protect L. lactis strain ATCC 19435 from H₂O₂.     

Involvement of Molecular Oxygen in the Enzyme-Catalyzed NADH Oxidation and Ferric Leghemoglobin Reduction

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb⁺³), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O₂ was removed from the reaction system, but all were restored upon readdition of O₂. In the absence of exogenous electron carriers and in the presence of O₂ and excess NADH, FLbR catalyzed NADH oxidation with the generation of H₂O₂ functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction. The formation of H₂O₂ during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H₂O₂. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction were not constant but changed with time and with concentrations of NADH and O₂ in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb⁺³ by reaction intermediates. A study of the affinity of FLbR for O₂ showed that the enzyme required at least micromolar levels of dissolved O₂ for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.     

The role of external carbonic anhydrase in the photosynthetic use of inorganic carbon in the deep-water alga Phyllariopsis purpurascens (Laminariales, Phaeophyta)

Mechanisms of inorganic carbon assimilation were investigated in the deep-water alga Phyllariopsis purpurascens (C. Agardh) Henry et South (Laminariales, Phaeophyta). The gross photosynthetic rate as a function of external pH, at a constant concentration of 2 mM dissolved inorganic carbon (DIC), decreased sharply from pH 7.0 to 9.0, and was not substantially different from 0 above pH 9.0. These data indicate that P. purpurascens is inefficient in the use of external HCO₃ ⁻ as a carbon source in photosynthesis. Moreover, the photosynthetic rate as a function of external DIC and the highest pH (9.01 ± 0.07) that this species can achieve in a closed system were consistent with a low capacity to use HCO₃ ⁻, in comparison to many other species of seaweeds. The role of external carbonic anhydrase (CA; EC 4.2.1.1) on carbon uptake was investigated by measuring both the HCO₃ ⁻-dependent O₂ evolution and the CO₂ uptake, at pH 5.5 and 8.0, and the rate of pH change in the external medium, in the presence of selected inhibitors of extra- and intracellular CA. Photosynthetic DIC-dependent O₂ evolution was higher at pH 5.5 (where CO₂ is the predominant form of DIC) than at pH 8.0 (where the predominant chemical species is HCO₃ ⁻). Both intra- and extracellular CA activity was detected. Dextran-bound sulfonamide (DBS; a specific inhibitor of extracellular CA) reduced the photosynthetic O₂ evolution and CO₂ uptake at pH 8.0, but there was no effect at pH 5.5. The pH-change rate of the medium, under saturating irradiance, was reduced by DBS. Phyllariopsis purpurascens has a low efficiency in the use of HCO₃ ⁻ as carbon source in photosynthesis; nevertheless, the ion can be used after dehydration, in the external medium, catalyzed by extracellular CA. This mechanism could explain why the photosynthetic rate in situ was higher than that supported solely by the diffusion of CO₂ from seawater. Received: 6 March 1998 / Accepted: 22 June 1998     

Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H⁺ extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O₂ uptake with a 4H⁺ to O₂ ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.     

Vanadate stimulates oxidation of NADH to generate hydrogen peroxide

Vanadate in the polymeric form of decavanadate, but not other forms, stimulated oxidation of NADH to NAD⁺ NADPH was also oxidized with comparable rates. This oxidation of NADH was accompanied by uptake of oxygen and generated hydrogen peroxide with the following stoichiometry: NADH + H⁺ + O₂ → NAD⁺ + H₂O₂. The reaction followed second-order kinetics. The rate was dependent on the concentration of both NADH and vanadate and increased with decreasing pH. The reaction had an obligatory requirement for phosphate ions. Esr studies in the presence of the spin trap dimethyl pyrroline N oxide indicated the involvement of Superoxide anion as an intermediate. The reaction was sensitive to Superoxide dismutase and other scavengers of superoxide anions.     

A nitrilotriacetic acid monooxygenase with conditional NADH-oxidase activity.

A tertiary amine monoxygenase from a Pseudomonas sp. was partially purified (35-fold) and characterized. In the presence of nitrilotriacetate (NTA), O₂, NADH, and Mn²⁺, the enzyme yielded two sets of products: iminodiacetate, glyoxylate, NAD⁺ and H₂O; or H₂O₂ and NAD⁺. Which set of products predominated was a function of enzyme concentration, ionic strength of solution, pH, and cation supplied. NTA functioned both as a modifiable substrate and as a stimulator of NADH oxidase activity. A requirement for preincubation with Mn²⁺ and NTA to eliminate enzyme hysteresis and the similar Km values for NTA and Mn²⁺ suggested that the substrate and metal were bound as a unit by the enzyme.     

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD<Superscript>+</Superscript> regeneration

Objectives

To find an efficient and cheap system for NAD⁺ regeneration

Results

A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K _m value for NADH of 10 μM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD⁺ regeneration was established. The system is attractive in that the O₂ consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD⁺ was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C

Conclusion

The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD⁺ regeneration.

     

Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis

An O₂ electrode system with a specially designed chamber for `whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O₂ concentrations on photosynthetic O₂ evolution. At pH = 5.5 and 20% O₂, cells grown in HCO₃⁻ medium (low CO₂, pH ≥ 9.0) exhibited a higher affinity for external CO₂ (K_½(CO₂) = 40 ± 6 micromolar) than the cells grown for at least 24 hours in high-CO₂ medium (pH = 6.5), (K_½(CO₂) = 94 ± 16 micromolar). With O₂ ≤ 2% in contrast, both types of cells showed a high apparent affinity (K_½(CO₂) = 50 − 52 micromolar). A Warburg effect was detectable only in the low affinity cells previously cultivated in high-CO₂ medium (pH = 6.5). The high-pH, HCO₃⁻-grown cells, when exposed to low pH (5.5) conditions, exhibited a response indicating an ability to fix CO₂ which exceeded the CO₂ externally supplied, and the reverse situation has been observed in high-CO₂-grown cells. At pH 8.2, the apparent photosynthetic affinity for external HCO₃⁻ (K_½[HCO₃⁻]) was 0.6 ± 0.2 millimolar, at 20% O₂. But under low O₂ concentrations (≤2%), surprisingly, an inhibition of net O₂ evolution was elicited, which was maximal at low HCO₃⁻ concentrations. These results indicate that: (a) photorespiration occurs in this alga and can be revealed by cultivation in high-CO₂ medium, (b) Chara cells are able to accumulate CO₂ internally by means of a process apparently independent of the plasmalemma HCO₃⁻ transport system, (c) molecular oxygen appears to be required for photosynthetic utilization of exogenous HCO₃⁻: pseudocyclic electron flow, sustained by O₂ photoreduction, may produce the additional ATP needed for the HCO₃⁻ transport.     

Effects of Polyamines on the Oxidation of Exogenous NADH by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria

The effect of polyamines (putrescine, spermine, and spermidine) on the oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L. cv. OB1) mitochondria, have been studied. Addition of spermine and/or spermidine to a suspension of mitochondria in a low-cation medium (2 millimolar-K⁺) caused a decrease in the apparent K_m and an increase in the apparent V_max for the oxidation of exogenous NADH. These polycations released by screening effect the mitochondrially induced quenching of 9-aminoacridine fluorescence, their efficiency being dependent on the valency of the cation (C⁴⁺ > C³⁺). Conversely, putrescine only slightly affected both kinetic parameters of exogenous NADH oxidation and the number of fixed charges on the membranes. Spermine and spermidine, but not putrescine, decreased the apparent K_m for Ca²⁺ from about 1 to about 0.2 micromolar, required to activate external NADH oxidation in a high-cation medium, containing physiological concentrations of Pi, Mg²⁺ and K⁺. The results are interpreted as evidence for a role of spermine and spermidine in the modulation of exogenous NADH oxidation by plant mitochondria in vivo.     

Induction by Oxygen of Respiration and Phosphorylation of Anaerobically Grown Escherichia coli

The changes occurring in the respiratory enzymes of anaerobically grown Escherichia coli strain B and E. coli 15 T⁻A⁻U⁻bar during exposure to oxygen were studied. Reduced nicotinamide adenine dinucleotide (NADH) oxidase activity reached its peak soon after O₂ exposure; cytochrome content and succinate oxidase activity increased more slowly, and these increases paralleled each other. The activities of isocitrate and malate dehydrogenases also increased, but the increase was less than that of the succinate and NADH oxidases; exposure to O₂ had no effect on the succinate and NADH dehydrogenase activities. On the other hand, the glycolytic activity decreased slowly after O₂ exposure. The incorporation of ³²P into acid-soluble organic phosphate esters paralleled the respiratory rate during the first 60 min after O₂ exposure, but continued to increase after the respiration reached a plateau. The sensitivity of ³²P incorporation to the uncoupler carbonyl cyanide m-chlorophenylhydrazone also increased with time. The observed relationship between the development of the respiratory chain and the energy-conserving mechanism during O₂ exposure is discussed. Synthesis of the respiratory enzymes upon exposure to oxygen was dependent on concomitant protein and ribonucleic acid synthesis but not on deoxyribonucleic acid synthesis.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

Energy-coupling mechanisms under aerobic and anaerobic conditions in autotrophically grown Pseudomonas saccharophila

The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H₂ or various other organic electron donors to either O₂ or NO₃^? with concomitant ATP generation. The respective $\text{P}\text{O}$ ratios with H₂ and NADH were 0.63 and 0.73, the respective $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios were 0.57 and 0.54. In contrast, the $\text{P}\text{O}$ and $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O₂ or NO₃^? as terminal electron acceptors without affecting the oxidation of H₂ or other substrates. The NADH oxidation at the expense of O₂ or NO₃^? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H₂ oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H₂, and succinate-linked electron transport to O₂ or NO₃^? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O₂ or NO₃^?, the energy conservation coupled to H₂ oxidation under aerobic or anaerobic conditions appeared to involve site II only.     

Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [¹⁴C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and ¹⁴CO₂ bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO₂ in the chloroplast.     

Decrease in membrane hydraulic conductance of rhizodermal cells under nitrate deficit is related to acidification at the root surface

Barley seedlings (Hordeum vulgare L.) were grown on porous plates submerged in Knop medium at pH 6.0 (control) and in a similar nutrient solution where NO ₃ ^? was replaced with Cl^? (treatment); in some treatments Mes buffer (10–20 mM, pH 6.0) was added to the medium. In the absence of buffer, the pH of the medium shifted towards the alkaline region in the presence of NO ₃ ^? and to the acidic region in the presence of Cl^?, with the total shift of no more than 0.3 pH units per day. The replacement of NO ₃ ^? with Cl^? (in a buffer-free medium) decreased the hydraulic membrane conductance of rhizodermal cells (L _p) within a 4-h period; after one day L _p settled at approximately 50% of its initial value observed in untreated plants. When the removal of nitrate from the medium was accompanied by the addition of buffer, no changes in L _p were observed over a 1-day period. The perfusion of external solution (at a rate of 10 mm/s) made it possible to control pH in the proximity to root surface (pH_s). These experiments showed that L _p was independent of the surface pH in the pH_s range 7.0–5.0, whereas at pH_s = 4.5 L _p decreased within 15 min to a steady-state level of about 50% of the control value. It is concluded that the reduction of L _p under nitrate deficit was related to acidification of the medium near the root surface. The acidic pH shift could be caused by the cessation of proton/nitrate symport and by activation of the plasmalemma H⁺-pump, related to changes in the cytosolic pH-stat.     

External Pathway of NADH Oxidation in the Liver of the River Lamprey Lampetra fluviatilis

A possibility of exogenous NADH oxidation via the external pathway has been shown on homogenates and isolated liver cells of the lamprey Lampetra fluviatilis in the presence of rotenone and antimycin A. The homogenates were incubated in isotonic and hypotonic sucrose media, while cells, in isotonic salt medium. At incubating the tissue preparations in isotonic media, digitonin was used to enhance membrane permeability to NADH and cytochrome c. In homogenates, the maximal rate of NADH oxidation via the external pathway in the presence of cytochrome c and digitonin was 5.3 nmol O₂/min/10 mg wet weight. This value in the cells amounted to 12.6, while without addition of exogenous NADH and cytochrome c, to 11.0 nmol O₂/min/10 million cells. Cyanide inhibited completely the NADH oxidation via the external pathway both in homogenates and in cells. The intact lamprey hepatocytes, unlike homogenates, are suggested to contain sufficient concentrations of cytochrome c and extramitochondrial NADH to provide maximal NADH oxidation rate in mitochondria through external pathway. This allows thinking that potential possibilities of NADH oxidation via the external pathway in Cyclostomata and mammals are qualitatively and quantitatively close.     

Na+-Ca2+ exchange activity in rabbit lymphocyte plasma membranes

Plasma membranes of rabbit thymus lymphocytes accumulated Ca²⁺ when a Na⁺ gradient (intravesicular > extravesicular) was formed across the membranes. Dissipation of the Na⁺ gradient by the addition of Na⁺ to the external medium decreased Ca²⁺ uptake. Ca²⁺ preloaded into the lymphocytes was extruded when Na⁺ was added to the external medium. The Ca²⁺ uptake decreased at acidic pH but increased at alkaline pH (above 8) and the activity was saturable for Ca²⁺ (apparent K_m for Ca²⁺ was 61 μM and apparent V_max was 11.5 nmol/mg protein per min). Na⁺-dependent uptake of Ca²⁺ was inhibited by tetracaine and verapamil, and partially inhibited by La³⁺. The uptake was not influenced by orthovanadate.     

Photosynthetic Response to Alkaline pH in Anabaena variabilis

The rate of O₂ evolution and alkalization of the medium in low CO_2⁻ grown Anabaena variabilis was observed as affected by the pH in the medium. Both rates are severely inhibited by pH values higher than 9.5, but the latter is more sensitive to this treatment. This finding, as well as the lag observed in alkalization of the medium, but not in O₂ evolution, following the addition of HCO₃⁻ indicates that the transport of HCO₃ and OH⁻ (or H⁺) are not compulsorily coupled. The inhibition of photosynthesis by strongly alkaline pH is attributed to an alteration of the internal pH and, hence, the rate of carboxylation. This conclusion is supported by data showing that the rate of O₂ evolution is affected by pH more strongly at saturating [HCO₃⁻] than at limiting [HCO₃⁻]. Also, the rate of O₂ evolution at saturating light intensity is affected by pH more strongly than is the initial slope of the curve against light intensity or the rate of dark respiration.