Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria.

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.     

Purification of the plant alternative oxidase from Arum maculatum: measurement, stability and metal requirement

We have purified plant alternative oxidase (AOX) protein from the spadices of thermogenic Arum maculatum (cuckoo pint) to virtual homogeneity. The obtained enzyme fraction exhibits a high specific activity, consuming on average 32 micromol oxygen min(-1) mg(-1), which is completely stable for at least 6 months when the sample is stored at -70 degrees C. This exceptionally stable AOX activity is inhibited approximately 90% (I(50) approximately 10 microM) by 8-hydroxyquinoline (8-OHQ) and also, although to a lesser extent, by other metal chelators such as o-phenanthroline, alpha,alpha'-dipyridyl and EDTA. When inhibited by 8-OHQ, AOX activity is fully restored upon addition of 1.2 mM ferric iron, but neither ferrous iron nor manganese has any effect, whilst zinc decreases activity even further. Furthermore, we have developed a spectrophotometric assay to measure AOX activity in an accurate manner, which will facilitate future steady state and transient kinetic studies. The reliability of this assay is evidenced by retained stability of AOX protein during the course of the reaction, reproducibility of the measured initial rates, an observed 2:1 duroquinol-oxygen stoichiometry and by the fact that, in absolute terms, the measured rates of duroquinone formation and duroquinol disappearance are identical.     

Iron-sulphur centres in mitochondria from Arum maculatum spadix with very high rates of cyanide-resistant respiration.

X-band electron-paramagnetic-resonance spectroscopy at 4.2--77K combined with measurements of oxidation-reduction potential was used to identify iron--sulphur centres in Arum maculatum (cuckoo-pint) mitochondria. In the oxidized state a signal with a derivative maximum at g = 2.02 was assigned to succinate dehydrogenase centre S-3. Unreduced particles showed additional signals at g = 2.04 and 1.98 (at 9.2 GHz), which may be due to a spin-spin interaction. In the reduced state a prominent signal at g = 1.93 and 2.02 was resolved into at least three components that could be assigned to centres S-1 and S-2 of succinate dehydrogenase (midpoint potentials -7 and -240 mV respectively at pH 7.2) and a small amount of centre N-1b (e'o= -240 mV) of NADH-ubiquinone reductase. In addition, changes in line shape around -10 mV indicated the presence of a fourth component in this signal. The latter was more readily reduced by NADH than by succinate, suggesting that it might be associated with the external NADH dehydrogenase. The iron-sulphur centres of NADH-ubiquinone reductase were present in an unusually low concentration, indicating that the alternative, non-phosphorylating, NADH dehydrogenase containing a low number of iron-sulphur centres may be responsible for most of the high rate of oxidation of NADH.     

Subcellular localization of two porphyrin-synthesis enzymes in Pisum sativum (pea) and Arum (cuckoo-pint) species.

The subcellular location of the two porphyrin-synthesis enzymes 5-aminolaevulinate dehydratase (ALAD) and porphobilinogen deaminase (PBGD) was investigated in Pisum sativum (pea) leaves and spadices of Arum (cuckoo-pint). Throughout the tissue-fractionation procedures the distribution of the two enzymes paralleled that of the plastid marker enzyme (ADP-glucose pyrophosphorylase), even in Arum, a tissue where the synthesis of non-plastid haem is predominant. The distribution of cytosolic marker enzyme (lactate dehydrogenase) was significantly different from that of ALAD and PBGD and, although purified mitochondria from both species had some residual activity, this was always less than contaminating plastid marker enzyme. The results suggest that ALAD and PBGD are exclusively plastid enzymes. The significance of this for the role of plastids in cellular porphyrin synthesis is discussed.     

Partial purification of the cyanide-resistant alternative oxidase of skunk cabbage (Symplocarpus foetidus) mitochondria.

A partial purification of the cyanide-resistant, alternative oxidase from skunk cabbage (Symplocarpus foetidus L.) spadix mitochondria is described. Skunk cabbage mitochondria were solubilized in N,N-bis-(3-D-glucon-amido-propyl)deoxycholamide and the alternative oxidase was purified using a batch DEAE-cellulose treatment, followed by precipitation with Extracti-Gel and chromatography on Sephadex G-200. Following pooling and concentrating of the most active fractions from the gel filtration column, a 20- to 30-fold purification of the alternative oxidase was obtained, with no evidence of contamination by cytochrome c oxidase (complex IV) or cytochrome c reductase (complex III). Polyacrylamide gel electrophoresis of the partially purified oxidase showed major polypeptides at 36 and 29 kD, both of which react with monoclonal antibodies raised against the Sauromatum guttatum alternative oxidase. The purified oxidase fraction showed no absorbance in the visible spectral region, and addition of sodium borohydride induced no absorbance changes in the ultraviolet region. The purified alternative oxidase catalyzed the four-electron reduction of oxygen to water in the absence of citrate, but catalyzed an apparent two-electron reduction of oxygen to hydrogen peroxide in the presence of 0.7 M citrate.     

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria

Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca²⁺ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH.     

Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.

Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.     

Stimulation of TRPC5 cationic channels by low micromolar concentrations of lead ions (Pb)

Lead toxicity is long-recognised but continues to be a major public health problem. Its effects are wide-ranging and include induction of hyper-anxiety states. In general it is thought to act by interfering with Ca²⁺ signalling but specific targets are not clearly identified. Transient receptor potential canonical 5 (TRPC5) is a Ca²⁺-permeable ion channel that is linked positively to innate fear responses and unusual amongst ion channels in being stimulated by trivalent lanthanides, which include gadolinium. Here we show investigation of the effect of lead, which is a divalent ion (Pb²⁺). Intracellular Ca²⁺ and whole-cell patch-clamp recordings were performed on HEK 293 cells conditionally over-expressing TRPC5 or other TRP channels. Extracellular application of Pb²⁺ stimulated TRPC5 at concentrations greater than 1 μM. Control cells without TRPC5 showed little or no response to Pb²⁺ and expression of other TRP channels (TRPM2 or TRPM3) revealed partial inhibition by 10 μM Pb²⁺. The stimulatory effect on TRPC5 depended on an extracellular residue (E543) near the ion pore: similar to gadolinium action, E543Q TRPC5 was resistant to Pb²⁺ but showed normal stimulation by the receptor agonist sphingosine-1-phosphate. The study shows that Pb²⁺ is a relatively potent stimulator of the TRPC5 channel, generating the hypothesis that a function of the channel is to sense metal ion poisoning.     

Measurements of starch breakdown as estimates of glycolysis during thermogenesis by the spadix of Arum maculatum L.

The rates of starch breakdown at thermogenesis by clubs of the spadices of Arum maculatum L. were measured in individual clubs still attached to the plants. The values obtained were used as estimates of the rate of glycolysis at thermogenesis. Such rates were shown to exceed the maximum catalytic activities of phosphofructokinase (E.C. 2.7.1.11.), aldolase (E.C. 4.1.2.7.), and glyceraldehydephosphate dehydrogenase (E.C. 1.2.1.12.) in developing clubs. The marked increases in the activities of the above enzymes that occur during the development of the club have been shown to be a prerequisite for the attainment of the high rate of glycolysis found at thermogenesis, and thus to be an example of coarse control of glycolysis.     

Induction and activation of the alternative oxidase of potato tuber mitochondria

Potato tubers ( Solanum tubersum L. cv. Grata) were stored for atleast 1 week at room temperature and then incubated with an equal amount of apples ( Malus domestica L.) for 2 days. After this treatment, intact tuber mitochondria isolated by Percoll gradient centrifugation showed a high degree of induction of the alternative oxidase, measured as cyanide-resistant, salicylhydroxamic acid-sensitive respiration. With succinate as substrate an activity of more than 130 nmol O₂(mg protein) ¹ min ^t was obtained. An assay of the alternative oxidase using duroquinol as an electron donor was developed. To become reliable the assay required the presence of defatted bovine serum albumin (BSA) and catalase (EC 1. 11. 1. 6). Furthermore, a lowering of the assay temperature to 15°C improved the stability of the duroquinol-based activity. One remarkable finding was that with duroquinol (or external NADH) as substrate the alternative oxidase was synergistically activated by succinate (as well as by malate) even in the presence of the succinate dehydrogenase inhibitor malonate. Our interpretation is that succinate and malate (indirectly) activate the alternative oxidase and that this activation is part of a physiological mechanism for regulation of the alternative oxidase.     

Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria.

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.     

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD⁺-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (–2 Arg changed to –2 Gly), the processing of the precursor was inhibited.     

The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria

In addition to possessing multiple NAD(P)H dehydrogenases, most plant mitochondria contain a cyanide- and antimycin-insensitive alternative terminal oxidase. Although the general characteristics of this terminal oxidase have been known for a considerable number of years, the mechanism by which it is regulated is unclear and until recently there has been relatively little information on its exact nature. In the past 5 years, however, the application of molecular and novel voltametric techniques has advanced our understanding of this oxidase considerably. In this article, we review briefly current understanding on the structure and function of the multiple NADH dehydrogenases and consider, in detail, the nature and regulation of the alternative oxidase. We derive a kinetic model for electron transfer through the ubiquinone pool based on a proposed model for the reduction of the oxidase by quinol and show how this can account for deviations from Q-pool behaviour. We review information on the attempts to isolate and characterise the oxidase and finally consider the molecular aspects of the expression of the alternative oxidase.     

Evidence for stabilising selection acting on flowering time in Arum maculatum (Araceae): the influence of phylogeny on adaptation

The relationship between flowering time and reproductive success was investigated in the fly-pollinated, monoecious perennial herb Arum maculatum L. (Araceae). This species temporarily traps its principle pollinator, a psychodid midge. Probability of fruit set was analysed in relation to early, peak and late periods of the flowering phenology of four British populations between 1992 and 1997. In three out of five cases, plants which flowered during early and late periods were significantly less likely to set fruit. In addition, one population showed a similar relationship for percentage fruit set of individual inflorescences, and seeds from peak-flowering plants were significantly heavier. There was no variation in number of female flowers per inflorescence over the flowering season. Probability of fruit set appears to be mediated by the likelihood of trapping psychodid midges that have previously been trapped and picked up pollen, an unlikely event during early and late flowering periods when few inflorescences are open. The majority of plants in all populations produce only one inflorescence which means that timing of flowering may be crucial to reproductive success. We interpret our findings as evidence that stabilising selection may be acting on some populations and/or during some years. The ultimate cause, however, can be related to the very short (12–18 h) female phase of each inflorescence, a phylogenetically conservative trait within the Araceae. Received: 19 August 1998 / 15 February 1999