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.     

Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase Activity in the Posterior Gills of the Blue Crab, <Emphasis Type="Italic">Callinectes ornatus</Emphasis> (Decapoda,Brachyura): Modulation of ATP Hydrolysis by the Biogenic Amines Spermidine and Spermine

We investigated the effect of the exogenous polyamines spermine, spermidine and putrescine on modulation by ATP, K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ and on inhibition by ouabain of posterior gill microsomal Na⁺,K⁺-ATPase activity in the blue crab, Callinectes ornatus, acclimated to a dilute medium (21‰ salinity). This is the first kinetic demonstration of competition between spermine and spermidine for the cation sites of a crustacean Na⁺,K⁺-ATPase. Polyamine inhibition is enhanced at low cation concentrations: spermidine almost completely inhibited total ATPase activity, while spermine inhibition attained 58%; putrescine had a negligible effect on Na⁺,K⁺-ATPase activity. Spermine and spermidine affected both V and K for ATP hydrolysis but did not affect ouabain-insensitive ATPase activity. ATP hydrolysis in the absence of spermine and spermidine obeyed Michaelis–Menten behavior, in contrast to the cooperative kinetics seen for both polyamines. Modulation of V and K by K⁺, Na⁺, NH₄ ⁺ and Mg²⁺ varied considerably in the presence of spermine and spermidine. These findings suggest that polyamine inhibition of Na⁺,K⁺-ATPase activity may be of physiological relevance to crustaceans that occupy habitats of variable salinity.     

Polyamine Uptake, Kinetics, and Competition among Polyamines and between Polyamines and Inorganic Cations

Polyamine uptake, the kinetics of this uptake, and the competition among polyamines and between polyamines and inorganic cations were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments using ¹⁴C-labeled polyamines were carried out on single petals, at room temperaure (20°C) and in the light. The results show that putrescine, spermidine, and spermine uptake was dependent on the external pH and occurred up to high external polyamine concentrations with K_m values of 8.6, 1.2, and 2.1 millimolar, respectively, with spermidine being the most absorbed at low concentration (17 micromolar). Putrescine and spermidine did not seem to compete for the same site of absorption. Furthermore, putrescine and spermidine uptake was not inhibited by Ca²⁺, Mg²⁺, and K⁺ at the same concentrations (17 micromolar), whereas 1.7 millimolar Ca²⁺ inhibited and K⁺ enhanced spermidine uptake. The intracellular localization of the absorbed putrescine was determined using two different methods. Very little label was found in the apoplast, while most of it was localized in the 98,500g supernatant. According to our data the vacuole, which represents a substantial part of Saintpaulia parenchyma cells, could be a site of putrescine accumulation. 2,4-Dinitrophenol and diethylstilbestrol did not inhibit uptake; however, at 0°C there was a 35% inhibition of spermidine uptake, compared with the controls kept at 20°C as well as a 68% inhibition with 20 millimolar NaSCN.     

What is the role of putrescine accumulated under potassium deficiency?

Biomarker metabolites are of increasing interest in crops since they open avenues for precision agriculture, whereby nutritional needs and stresses can be monitored optimally. Putrescine has the potential to be a useful biomarker to reveal potassium (K⁺) deficiency. In fact, although this diamine has also been observed to increase during other stresses such as drought, cold or heavy metals, respective changes are comparably low. Due to its multifaceted biochemical properties, several roles for putrescine under K⁺ deficiency have been suggested, such as cation balance, antioxidant, reactive oxygen species mediated signalling, osmolyte or pH regulator. However, the specific association of putrescine build-up with low K⁺ availability in plants remains poorly understood, and possible regulatory roles must be consistent with putrescine concentration found in plant tissues. We hypothesize that the massive increase of putrescine upon K⁺ starvation plays an adaptive role. A distinction of putrescine function from that of other polyamines (spermine, spermidine) may be based either on its specificity or (which is probably more relevant under K⁺ deficiency) on a very high attainable concentration of putrescine, which far exceeds those for spermidine and spermine. putrescine and its catabolites appear to possess a strong potential in controlling cellular K⁺ and Ca²⁺, and mitochondria and chloroplasts bioenergetics under K⁺ stress.     

Oxidation of spermine by an amine oxidase from lentil seedlings

Spermine is a substrate of lentil (Lens culinaris) seedling amine oxidase and the oxidation products are reversible inactivators of the enzyme. The spermine is oxidized at the terminal amino groups to a dialdehyde: 2 moles of hydrogen peroxide and 2 moles of ammonia per mole of spermine are formed. The pH optimum of the enzyme with spermine is 7.9 in TI-HCI buffer; the K_m value is 4.4·10⁻⁴ molar, similar to that found with other substrates (putrescine and spermidine).     

Modulation of nutrient acquisition and polyamine pool in salt-stressed wheat (Triticum aestivum L.) plants inoculated with arbuscular mycorrhizal fungi

Two wheat (Triticum aestivum L.) cultivars, Sids 1 and Giza 168, were grown under non-saline or saline conditions (4.7 and 9.4 dS m^?1) with and without arbuscular mycorrhizal fungi (AMF) inoculation. Salt stress considerably decreased root colonization, plant productivity and N, P, K⁺, Fe, Zn and Cu concentrations, while it increased Na⁺ level, particularly in Giza 168. Mycorrhizal colonization significantly enhanced plant productivity and N, P, K⁺, Fe, Zn and Cu acquisition, while it diminished Na⁺ uptake, especially in Sids 1. Salinity increased putrescine level in Giza 168, however, values of spermidine and spermine increased in Sids 1 and decreased in Giza 168. Mycorrhization changed the polyamine balance under saline conditions, an increase in putrescine level associated with low contents of spermidine and spermine in Giza 168 was observed, while Sids 1 showed a decrease in putrescine and high increase in spermidine and spermine. Moreover, mycorrhizal inoculation significantly reduced the activities of diamine oxidase and polyamine oxidase in salt-stressed wheat plants. Modulation of nutrient acquisition and polyamine pool can be one of the mechanisms used by AMF to improve wheat adaptation to saline soils. This is the first report dealing with mycorrhization effect on diamine oxidase and polyamine oxidase activities under salt stress.     

Effect of Exogenous Putrescine, Spermidine, and Spermine on K Uptake and H Extrusion through Plasmamembrane in Maize Root Segments

The action of exogenous polyamines (putrescine, spermidine, and spermine) on `washing' and fusicoccin-stimulated K⁺ uptake and H⁺ extrusion through the plasmamembrane in maize (Zea mays L., hybrid line Plenus S 516) root apical segments was studied. The results showed that polyamines inhibit the washing-stimulated K⁺ influx and H⁺ extrusion without interfering with K⁺ uptake and H⁺ extrusion stimulated by fusicoccin. Spermidine appeared to be the most effective in inhibiting K⁺ uptake and H⁺ extrusion while putrescine showed a smaller inhibiting action with respect to the others. The analysis of kinetic constants indicated that the polyamines behave as competitive inhibitors with respect to K⁺.     

Glyceollin: a site-specific inhibitor of electron transport in isolated soybean mitochondria

The glyceollin inhibition of electron transport by isolated soybean and corn mitochondria was similar to that of rotenone, acting at site I between the internal NADH dehydrogenase and coenzyme Q. Coupled state 3 malate oxidation was inhibited by glyceollin and rotenone with apparent K_i values of about 15 and 5 micromolar, respectively. Carbonylcyanide m-chlorophenyl hydrazone uncoupled state 4 malate oxidation was also inhibited by glyceollin and rotenone, but uncoupled succinate and exogenous NADH state 4 oxidation was only slightly inhibited by both compounds. Glyceollin also inhibited ferricyanide reduction with malate as the electron donor, with an apparent K_i of 5.4 micromolar, but failed to inhibit such reduction with succinate or externally added NADH as electron donors. Glyceollin did not inhibit state 4 oxidation of malate, succinate, or exogenous NADH. Glyceollin did not act as a classical uncoupler or as an inhibitor of oxidative phosphorylation.     

Cytoplasmic polyamines block the fast-activating vacuolar cation channel

The fast-activating vacuolar (FV) channel dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Since polyamines are known to increase in plant cells in response to stress, the regulation of FV channels by polyamines was investigated. Patch-clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. The trivalent polyamine spermidine and the tetravalent polyamine spermine blocked FV channels with K_d≈ 100 μM and K_d≈ 5 μM, respectively. Increasing cytosolic and vacuolar Ca²⁺ had no effect on putrescine and spermidine binding to FV channels but slightly decreased the affinity for spermine. The inhibition of FV channels by all three polyamines was not voltage-dependent. This points to a different mode of binding compared to inward rectifier K⁺ channels and Ca²⁺-permeable glutamate receptor channels from animal cells, which show rectification due to a voltage-dependent block by polyamines. In plant cells, the common polyamines (putrescine, spermidine and spermine) are likely to mediate a salt stress-induced decrease of ion flux across the vacuolar membrane by blocking FV channels.     

Kinetic Properties of Fungal Polyamine Oxidases and Their Application to Differential Determination of Spermine and Spermidine

Polyamine oxidase from Penicillium chrysogenum oxidized spermine rapidly and spermidine slightly at pH 7.5. The apparent Km values for spermine and spermidine were calculated to be 2.25 × 10^?5 m and 9.54 × 10^?6 m, respectively. The relative maximum velocities for spermine and spermidine were 3.37 × 10^?3 m (H₂O₂) per min per mg of protein and 2.08 × 10^?4 m (H₂O₂) per min per mg of protein, respectively. Spermine oxidation of the enzyme was competitively inhibited by spermidine and putrescine. The apparent Ki values by spermidine and putrescine were calculated to be 3.00 × 10^?5 m and 1.80 × 10^?8 m, respectively. On the other hand, polyamine oxidase from Aspergillus terreus rapidly oxidized both spermidine and spermine at pH 6.5. The apparent Km values for spermidine and spermine were 1.20 × 10^?8 m and 5.37 × 10^?7 m, respectively. The relative maximum velocities for spermidine and spermine were 1.55 × 10^?2 m (H₂O₂) per min per mg of protein and 6.20 × 10^?3 m (H₂O₂) per min per mg of protein, respectively.

Differential determination of spermine and spermidine was carried out using the two enzymes. The initial rate was assayed with Penicillium enzyme and the end point was measured afte addition of Aspergillus enzyme. Small amounts of polyamines (25 to 200 nmol of spermine and 25 to 250 nmol of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in close agreement with those obtained by an amino-acid analyzer.     

Purification and properties of agmatine iminohydrolase from groundnut cotyledons

Agmatine iminohydrolase was purified ca 375-fold from groundnut cotyledons. The enzyme exhibited an optimum pH between 5.5 and 8.5 and the energy of activation was 22 kcal/mol. The K_m for agmatine was (7.57 ± 0.77) × 10^?4 M. The enzyme was inhibited by tryptamine, putrescine, cadaverine, spermidine and spermine. Inhibition by cadaverine and spermidine was competitive. The K_i values for cadaverine and spermidine were 4.1 × 10^?3 and 7.5 × 10^?4 M, respectively.     

Response of grape rootstocks to salinity: changes in root growth, polyamines and abscisic acid

Effects of salinity (0, 50, 100 and 250 mM NaCl) on growth, root:shoot dry mass ratio, osmotic potential (ψ_x), electrolyte leakage and contents of Na⁺ and K⁺, polyamines and abscisic acid (ABA) were studied in the grape rootstocks Dogridge, 1613, St. George and Salt Creek. In control rootstocks, the root length was highest in Dogridge and contents of K⁺ and ABA in Salt Creek. Salinity treatments increased root Na⁺ and decreased K⁺ content and St. George exhibited highest Na⁺ content and Na⁺:K⁺ ratio. The root:shoot dry mass ratio in all rootstocks increased upto 100 mM NaCl. With increasing NaCl concentration, putrescine, spermine and spermidine contents showed consistent increase and putrescine increase was highest in St. George and spermidine and spermine in the Dogridge and Salt Creek. Under salinity, the ABA content increased in all the rootstocks but more in Salt Creek and Dogridge than in St. George.     

Changes in growth,photosynthetic capacity and ionic relations in spring wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) due to pre-sowing seed treatment with polyamines

The influence of pre-sowing seed treatment with polyamines (2.5 mM putrescine, 5.0 mM spermidine and 2.5 mM spermine) on growth, photosynthetic capacity, and ion accumulation in two spring wheat (Triticum aestivum L.) cultivars MH-97 (intolerant) and Inqlab-91 (tolerant) was examined. The primed seeds of each treatment and non-primed seeds were sown in a field containing 15 dS m⁻¹ NaCl. Although all three polyamines were effective in improving shoot growth and grain yield in both cultivars under saline conditions, the effect of spermine was very pronounced particularly in improving grain yield. Different priming agents did not affect the net CO₂ assimilation rate and transpiration rate of either cultivar. However, pre-treatment with spermidine increased stomatal conductance (g_s) in the tolerant cultivar, whereas with spermine stomatal conductance decreased in the intolerant cultivar under salt stress. Priming agents had different effects on the accumulation of different ions in wheat plant tissues. When spermidine and distilled water were used as priming agents, they were effective in reducing shoot [Na⁺] in the tolerant and intolerant cultivars, respectively under saline conditions. Although all priming agents caused an increase in shoot [K⁺], distilled water was more effective in improving shoot [K⁺] in both cultivars under salt stress. Pre-treatment with spermidine was very effective in reducing shoot [Cl⁻] under saline conditions particularly in the tolerant cultivar. However, the pattern of accumulation of different ions in roots due to different seed priming treatments was not consistent in either cultivar except that root Na⁺ decreased due to priming with spermine and spermidine in the intolerant and tolerant cultivars under saline conditions. In conclusion, although all three priming agents, spermine, spermidine and putrescine, were effective in alleviating the adverse effect of salt stress on wheat plants, their effects on altering the concentration of different ions and growth were different in the two cultivars differing in salt tolerance.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis

Addition of NAD⁺ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O₂ uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD⁺ (NAP₄-NAD⁺), an inhibitor of NAD⁺ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD⁺-stimulated malate-cytochrome c reductase activity, and reduction of added NAD⁺ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD⁺ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD⁺ by storing on ice for 72 hours, was completely dependent on added NAD⁺, and the effect of NAD⁺ on these mitochondria was prevented by incubating them with NAP₄-NAD⁺. External NAD⁺ reduction by these mitochondria was not affected by NAP₄-NAD⁺. We conclude that all effects of exogenous NAD⁺ on plant mitochondrial respiration can be attributed to net uptake of the NAD⁺ into the matrix space.     

Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O₂ uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K⁺ permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V_max, probably reflecting a diffusion-limited process.     

Plant pyruvate dehydrogenase complex: analysis of the kinetic properties and metabolite regulation

The pyruvate dehydrogenase complex was isolated from the mitochondria of broccoli florets and shown to be similar in its reaction mechanism to the complexes from other sources. Three families of parallel lines were obtained for the initial velocity patterns, indicating a multisite ping-pong mechanism. The apparent K_m values obtained were 321 ± 18, 148 ± 13, and 7.2 ± 0.51 μm for pyruvate, NAD⁺, and CoA, respectively. Product inhibition studies using acetyl-CoA and NADH yielded results which were in agreement with those predicted by the multisite ping-pong mechanism. Acetyl-CoA and NADH were found to be competitive inhibitors versus CoA and NAD⁺, respectively. All other substrate-product combinations showed uncompetitive inhibition patterns, except for acetyl-CoA versus NAD⁺. Among various metabolites tested, only hydroxypyruvate (K_i = 0.11 mM) and glyoxylate (K_i = 3.27 mM) were found to be capable of inhibiting the broccoli enzyme to a significant degree. Initial velocity patterns using Mg²⁺? or Ca²⁺-thiamine pyrophosphate and pyruvate as the variable substrate were found to be consistent with an equilibrium ordered mechanism where Mg? or Ca-thiamine pyrophosphate bind first, with dissociation constants of 33.8 and 3 μm, respectively. The Mg- or Ca-thiamine pyrophosphate complexes also dissociated rapidly from the enzyme complex.     

Replacement of Mg2+ by polyamines in the aminoacylation of tRNA from Phaseolus

The ability of putrescine, spermidine and spermine to replace Mg²⁺ ions in the charging reaction of tRNA was estimated for seventeen amino acids. The polyamines promoted only the transfer reaction in the case of Leu, Ile, Val, Tyr and Arg. A synergistic effect was observed when spermine was added to a suboptimal concentration of Mg²⁺ (charging at only 5% of the optimal level). This synergistic effect was not observed for Ala, Asp-NH₂, His, Lys and Ser. Kinetic studies showed a slower aminoacylation rate in those experiments when spermine and Mg⁺² (at 5% of the Mg²⁺ optimal concn) were used together than with Mg²⁺ (at the optimal concn) alone.     

Putrescine-oxidase activity in adult bovine serum and fetal bovine serum

Summary Putrescine-oxidase activity was found in fetal bovine serum (FBS) with a pH optimum of 8.0 and in adult bovine serum (ABS) with a pH optimum of 9.8. The crude FBS enzyme had a K_M for putrescine of 2.58×10⁻⁶ m and a V_max of 0.53 nmol per hr per 50 μl serum. Aminoguanidine competitively inhibited the enzyme with a K_I of 1.8×10⁻⁸ m. Spermidine and spermine proved competitive inhibitors of putrescine for both the FBS and the crude ABS putrescine oxidases. The V_max for the ABS putrescine oxidase was 2.10 nmol per hr per 50 μl serum, and the K_M for putrescine, 50.3×10⁻⁶ m. The K₁ of the ABS putrescine oxidase for aminoguanidine was 41×10⁻⁶ m. On the basis of both the K_M and K_I values, the adult serum enzyme, at its optimal pH of 9.8, bound spermidine and spermine more avidly than the smaller putrescine and aminoguanidine; whereas the FBS enzyme, at pH 8.0, bound aminoguanidine and putrescine more tightly than the larger polyamines. Each of the enzymes retained over 80% of its activity after heating at 56°C for 30 min. Applications of these data to the study of polyamines in tissue culture and to the purification of diamine oxidases are discussed. This work was supported in part by a grant from the Cystic Fibrosis Foundation.     

Synthesis and Content of Polyamines in Bloodstream Trypanosoma brucei*

SYNOPSIS. The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [³H]ornithine, [¹⁴C]arginine and [¹⁴C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [¹⁴C]methionine. Putrescine and spermidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous ¹⁴C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.