Pyridine nucleotide transhydrogenase activity of soluble cardiac NADH dehydrogenase and particulate NADH-ubiquinone reductase

Pyridine nucleotide transhydrogenase activities of a highly purified soluble NADH dehydrogenase and particulate NADH-ubiquinone reductase (Complex I) differ in their pH optima (5.0 and 6.0, respectively) and in their sensitivity to inhibition by Mg²⁺ and ATP. The oxidation of NADPH with ferricyanide as acceptor is very similar in both preparations with a pH optimum around 5.0. It is concluded that Complex I possesses two types of transhydrogenase activity, whereas only one has been found in the soluble dehydrogenase.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Oxidation of External NAD(P)H by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria : A Kinetic and Inhibitor Study

The functional interaction between the externally located NAD(P)H dehydrogenase and the Q-pool acceptor site(s) in Percoll-purified mitochondria from Jerusalem artichoke (Helianthus tuberosus L. cv OB1) mitochondria has been investigated. Oxidation of exogenous NADH is stimulated by ubiquinone (UQ₁) with a parallel decrease of the apparent K_m for NADH. In the presence of saturating amounts of UQ₁ as electron acceptor, the K_m (NADH) is not affected by variations of the ionic strength. Conversely, the K_m for UQ₁ is decreased by the screening effect of negative charges on the outer membrane surface. Under low-ionic strength, the hydroxyflavone platanetin progressively inhibits NADH oxidation with a mean inhibition dose of approximately 3 nanomoles of inhibitor per milligram of protein. Interestingly, under high-ionic strength, oxidation of NADH proceeds through two platanetin binding sites, one of which has a lower affinity for the inhibitor (mean inhibition dose = 20 nanomoles per milligram protein), because it is located near the outer surface of the membrane. This latter site is the one involved in the oxidation of external NADPH and, possibly, also affected by spermine and spermidine. Similarly to NADH, oxidation of NADPH is fully sensitive to micromolar concentrations of free Ca²⁺ ions; in addition, similar concentrations of the sulfhydryl reagent mersalyl are required to inhibit both NADH and NADPH oxidative activities. The results are interpreted as evidence for the presence of a single nonspecific NAD(P)H dehydrogenase.     

DNA-dependent RNA polymerases from murine testis. Properties of two activities.

Multiple forms of DNA-dependent RNA polymerase activities have been isolated from nuclei of mouse testis. Using highly purified nuclei, two activities can be solubilized and are separable by DEAE-Sephadex chromatography; peak I eluting at 0.11–0.14 M and peak II eluting at 0.24–0.27 M (NH₄)₂SO₄. A third form of RNA polymerase activity is observed eluting at 0.31–0.33 M (NH₄)₂SO₄ when an extract from a less highly purified nuclear preparation is analysed. At concentrations of 0.125 μg/ml, peak I is insensitive to the toxin α-amanitin, peak II is totally inhibited, and peak III is partially inhibited. Peak I activity is optimal at pH 8.4 in the presence of Mg²⁺ (2–6 mM) or Mn²⁺ (1 mM) and uses native and heat-denatured DNA template equally well. Peak II has optimal activity at pH 7.9 in the presence of Mn²⁺ (2 mM) and heat-denatured DNA. Mg²⁺ has little effect on the activity of peak II.     

Regulation by Magnesium of Potato Tuber Mitochondrial Respiratory Activities

Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg²⁺. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg²⁺ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg²⁺. However, respiration of malate, citrate, and -ketoglutarate requires at least 4-mM Mg²⁺ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg²⁺ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or -ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg²⁺. Respiration of succinate is active without external and matrix Mg²⁺, although stimulated by the cation. Respiration of -ketoglutarate was strictly dependent on external Mg²⁺ required for substrate transport into mitochondria, and internal Mg²⁺ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg²⁺ but, unlike -ketoglutarate, some activity still remains without external Mg²⁺. All the substrates revealed insensitive to external and internal mitochondrial Ca²⁺, except the exogenous NADH dehydrogenase, which requires either external Ca²⁺ or Mg²⁺ for detectable activity. Calcium is more efficient than Mg²⁺, both having cumulative stimulation. Unlike Ca²⁺, Mn²⁺ could substitute for Mg²⁺, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg²⁺.     

Interactions between magnesium ions,pH, glucose-6-phosphate,and NADPH/NADP+ ratios in the modulation of chloroplast glucose-6-phosphate dehydrogenase in vitro

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from spinach chloroplasts is strongly affected by interactions between Mg²⁺, proton, and substrate concentrations. Mg²⁺ activates the enzyme to different degrees; however, it is not essential for enzyme activity. The Mg²⁺-dependent activation follows a maximum curve, magnitude and position of the maximum being dependent on pH and NADPH/NADP⁺ ratios. At a ratio of zero and pH 7.2, maximum activity is observed at 10 mM Mg²⁺. Increasing the NADPH/NADP⁺ ratio up to 1.7 (a ratio measured in the stroma during a light period), maximum activity is shifted to much lower Mg²⁺ concentrations. At pH 8.2 (corresponding to the pH of the stroma in the light) and at a high NADPH/NADP⁺ ratio, enzyme activity is not affected by the Mg²⁺ ion. The results are discussed in relation to dark-light-dark regulation of the oxidative pentose phosphate cycle in spinach chloroplasts.Abbreviations DTT dithiothreitol - G-6-P glucose-6-phosphate - G-6-PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - PPC pentose phosphate cycle     

NAD(P)H-ubiquinone oxidoreductases in plant mitochondria

Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca²⁺ with aK _0.5 of about 1 µM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca²⁺ with aK _0.5 of 3 µM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer.     

Platanetin: A Potent Natural Uncoupler and Inhibitor of the Exogenous NADH Dehydrogenase in Intact Plant Mitochondria

Platanetin is a 3,5,7,8-tetrahydroxy, 6-isoprenyl flavone isolated from the bud scales of the plane tree (Platanus acerifolia Willd.). Its effects on the oxidative activities of isolated potato and mung bean mitochondria have been studied. The most noticeable effect is the selective inhibitory effect of this compound on the activity of the external NADH dehydrogenase of the inner membrane. A 50% inhibition of the NADH oxidation rate is obtained at a 2 micromolar concentration. This activity is probably due to the flavonoid structure and the high lipophilicity of platanetin associated with the presence of the isoprenyl chain. Another important effect of platanetin is its uncoupling activity on oxidative phosphorylation. The presence of easily dissociable hydroxyl groups and the high lipophilicity of platanetin allow a potent H⁺ transfer through the mitochondrial inner membrane. This uncoupling activity is comparable to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Platanetin is therefore the most active natural uncoupler known at the present time (full uncoupling at 2 micromolar with succinate as substrate). At higher concentrations (10 micromolar and more), platanetin can transfer electrons from the mitochondrial inner membrane to O₂; the branching point of this KCN-salicylhydroxamic acid insensitive platanetin dependent oxidative pathway is located at the level of flavoproteins, no transfer occurring when succinate is the substrate. The redox properties of platanetin are in accord with such an activity.     

Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

Regulation of the Na+/Ca2+ Exchanger by Pyridine Nucleotide Redox Potential in Ventricular Myocytes

The cardiac Na⁺/Ca²⁺ exchanger (NCX) is the major Ca²⁺ efflux pathway on the sarcolemma, counterbalancing Ca²⁺ influx via L-type Ca²⁺ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca²⁺ load and can contribute to abnormal Ca²⁺ handling and arrhythmias. NADH/NAD⁺ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD⁺ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (I_NCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced I_NCX inhibition was abolished by the H₂O₂ scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because I_NCX was insensitive to cytosolic NADPH, and NADH-induced ROS and I_NCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD⁺ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca²⁺ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases.     

Properties of substantially chlorophyll-free pea leaf mitochondria prepared by sucrose density gradient separation

Mitochondria isolated from pea leaves (Pisum sativum L. var Massey Gem) and purified on a linear sucrose density gradient were substantially free of contamination by Chl and peroxisomes. They showed high respiratory rates and good respiratory control and ADP/O ratios. Malate, glutamate, succinate, glycine, pyruvate, α-ketoglutarate, NADH, and NADPH were oxidized but little or no oxidation of citrate, isocitrate, or proline was detected. The oxidation of NADPH by the purified mitochondria did not occur via a transhydrogenase or phosphatase converting it to NADH. NADPH oxidation had an absolute requirement for added Ca²⁺, whereas NADH oxidation proceeded in its absence. In addition, oxidation of the two substrates showed different sensitivities to chelators and sulfhydryl reagents, and faster rates of O₂ uptake were observed with both substrates than with either alone. This indicates that the NADPH dehydrogenase is distinct from the exogenous NADH dehydrogenase.     

The internal rotenone‐insensitivae NADPH dehydrogenase contributes to malate oxidation by potato tuber and pea leaf mitochondria

Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, ND_in(NADPH) catalyzes the rotenone-insensitive and Ca²⁺-dependent oxidation of NADPH and ND_in(NADH) catalyzes the rotenone-insensitive and Ca²⁺-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, ND_in(NADPH) and ND_in (NADH) activity with a K_i of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in K_i between the two ND_in made possible the use of DPI inhibition to estimate ND_in (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O₂ mg^?1 min^?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD⁺ or NADP⁺) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP⁺ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of ND_in(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria.     

Pyruvate Dehydrogenase Complex from Chloroplasts of Pisum sativum L

Pyruvate dehydrogenase complex is associated with intact chloroplasts and mitochondria of 9-day-old Pisum sativum L. seedlings. The ratio of the mitochondrial complex to the chloroplast complex activities is about 3 to 1. Maximal rates observed for chloroplast pyruvate dehydrogenase complex activity ranged from 6 to 9 micromoles of NADH produced per milligram of chlorophyll per hour. Osmotic rupture of pea chloroplasts released 88% of the complex activity, indicating that chloroplast pyruvate dehydrogenase complex is a stromal complex. The pH optimum for chloroplast pyruvate dehydrogenase complex was between 7.8 and 8.2, whereas the mitochondrial pyruvate dehydrogenase complex had a pH optimum between 7.3 and 7.7. Chloroplast pyruvate dehydrogenase complex activity was specific for pyruvate, dependent upon coenzyme A and NAD and partially dependent upon Mg²⁺ and thiamine pyrophosphate.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

Kinetic and functional characterization of a membrane-bound NAD(P)H dehydrogenase located in the chloroplasts of Pleurochloris meiringensis (Xanthophyceae)

Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q₀, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K _m-values for both pyridine nucleotides were in the molar range (K _m[NADH]=9.8 M, K _m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP⁺ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP⁺ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme Q₀-2,3-dimethoxy-5-methyl-1,4-benzoquinone - FNR ferredoxin: NADP⁺ reductase - MD menadione - MV methylviologen - NDH NAD(P)H dehydrogenase - PQ plastoquinone - PQ₁₀ decylplastoquinone - SDH succinate dehydrogenase - UQ₁₀ decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone)     

Differential effects of ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonucleaw activity in non-apoptotic HL-60 promydocytic leukemia cell nuclei cleaved the chromatin of he autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL-60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca²⁺. but not by exogenous Mg²⁺. In Ca²⁺/Mg²⁺-free nuclei digation buffer, addition of Ca^2→ (1-10 mmol/L) induced endonuclease activity in the isolated nuclei, while addition of Mg²⁺ had no effect. In the presence of Ca²⁺(0.1 mmol/L), endonuclease activity was enhanced by exogenous Mg²⁺ (0.1-10mmol/L). These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL-60 cells during apoptosis is activated by Ca²⁺ and further modulated by Mg²⁺ in the presence of ca²⁺.     

Purification and characterization of a psychrophilic glutathione reductase from Antarctic ice microalgae <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. Strain ICE-L

A psychrophilic glutathione reductase from Antarctic ice microalgae Chlamydomonas sp. Strain ICE-L was purified by ammonium sulfate fractionation and three steps of chromatography. The yield was up to 25.1% of total glutathione reductase in the crude enzyme extract. The glutathione reductase activity was characterized by the spectrophotometric method under different conditions. Purified glutathione reductase was separated by SDS-PAGE, which furnished a homogeneous band. The native molecular mass of the enzyme was 115 kDa. Apparent Km values for NADPH and NADH (both at 0.5 mmol L⁻¹ oxidized glutathione) were 22.3 and 83.8 μmol L⁻¹, respectively. It was optimally active at pH 7.5, and it was stable from pH 5 to 9. Its optimum temperature was 25°C, with activity at 0°C 23.5% of the maximum. Its optimum ion strength and optimum Mg²⁺ were 50–90 and 7.5 mmol L⁻¹, respectively. Ca²⁺, Mg²⁺, and cysteine substantially increased the activity of the enzyme but chelating agents, heavy metals (Cd²⁺, Pb²⁺, Cu²⁺, Zn²⁺, etc.), NADPH, and ADP had significant inhibitory effects. This glutathione reductase can be used to study the adaptation and mechanism of catalysis of psychrophilic enzymes, and it has a high potential as an environmental biochemical indicator under extreme conditions.     

The modulating effects of pH and benzyladenine on the Ca2+– and Mg2+-ATPases in the plasmalemma of wheat root cells

Plant cells frequently and rapidly have to respond to environmental changes for survival. Regulation of transport and other energy-requiring processes in the plasmalemma of root cells is therefore one important aspect of the ecological adaptation of plants. Wheat (Triticum aestivum L. cv. Drabant) was grown hydroponically, with or without 50 nM benzyladenine in the medium, and plasma membranes from root cells of 8-day-old plants were prepared by aqueous polymer two-phase partitioning. The influence of Ca²⁺ and Mg²⁺ on the plasmalemma ATPase activities was investigated. The presence of benzyladenine during growth increased the ATPase activity, that dependent upon Ca²⁺ more than that elicited by Mg²⁺. As a general characteristic, ATP was the preferred substrate, but all nucleotide tri- and diphosphates could be accepted with activities in plasma membranes from control plants of 7-36% (Mg²⁺) and 40-86% (Ca²⁺) and in plasma membranes from benzyladenine-treated plants of 12-47% (Mg²⁺) and 53-102% (Ca²⁺) as compared with activities obtained with ATP. Nucleotidemonophosphates were not hydrolyzed by the preparations. In preparations from benzyladenine-treated plants one peak of Ca²⁺-ATPase at pH 5.2–5.6, with a tail from pH 6 and upwards, and one peak of Mg²⁺-ATPase at pH 6.0–6.5 were observed in the presence of EDTA in the assay media. In preparations from control plants, the addition of EDTA to the assays resulted in a wide optimum between pH 6 and 7 for Mg²⁺-ATPase and low Ca²⁺-ATPase activity with no influence of pH in the range 4.5 to 8. Analysis of the pH dependence in the presence of both Ca²⁺ and Mg²⁺ indicates that the control plants mainly contain Mg²⁺-ATPase corresponding to the proton pump. Preparations from benzyladenine-treated wheat roots show, in addition, activation by Ca²⁺, which, in the slightly alkaline pH range may correspond to a Ca²⁺-extruding (Ca²⁺+ Mg²⁺)-ATPase. In the acidic range, the responses are more complicated: the Mg²⁺-ATPase is inhibited by vanadate, while the Ca²⁺-ATPase is insensitive, and benzyladenine added during growth influences the interaction between Ca²⁺ and Mg²⁺ in a way that parallels the effect of high salt medium.     

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.     

The pH Dependence and the Binding Equilibria of the Calcium Indicator — Arsenazo III

The underlying principles of binding equilibria of arsenazo III with Ca²⁺ and Mg²⁺ are presented. Ca²⁺ and Mg²⁺ can bind arsenazo III in several different protonated forms depending on pH. The binding affinities of these different protonated forms of arsenazo III with Ca²⁺ increase in the order of H₄A_4- <H₃A^5- >H₂A^6- and with Mg²⁺, H₄A^4- > H₃A^5- > H₂A^6-. Arsenazo III is not membrane bound. The sensitivity ratio of arsenazo III with Ca²⁺ to arsenazo III with Mg²⁺ is close to two orders of magnitude. Arsenazo III and its complexes are extremely sensitive to pH changes. With 5 μM arsenazo III, the minimum detectable amount of Ca²⁺ can be as low as 0.08 μM. Contrary to current belief, we found that Mg²⁺ can bind to arsenazo III in a slightly acidic medium. Potential applications of arsenazo III to the study of membrane Ca²⁺ transport are also discussed.