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.     

Effects of Temperature on Electron Transport in Arum maculatum Mitochondria

The effects of temperature upon the respiratory pathways of Arum maculatum mitochondria have been studied. The alternate oxidase sustained a greater proportion of the total respiration at low temperatures than at higher temperatures. Arrhenius plots of respiratory activities show two discontinuities, one at 14°C and one at 21°C. The lower temperature discontinuity was associated with electron transport from succinate dehydrogenase to the alternative oxidase, enzymes that face the inner side of the membrane while the higher temperature discontinuity was associated with electron transport from the external NADH dehydrogenase to cytochrome c oxidase, which face the outer side of the membrane. Both discontinuities resulted in a decrease in the activation energy for electron transport on one side of the membrane. Arrhenius plots of transmembrane electron transport showed discontinuities at both 14° and 21°C but the upper discontinuity resulted in an increase in the activation energy. Activation energies determined for the respiratory activities show that above 21°C the exogenous NADH-cytochrome pathway and the succinate-alternative oxidase pathway were lower than those for the NADH-alternative pathway or the succinate cytochrome pathway.     

Succinoxidase and Cytochrome Oxidase in Mitochondria from the Spadix of Arum

1. Mitochondria prepared from young Arum spadix have enoughcytochrome c oxidase to account for the rate at which succinateis oxidized, but succinoxidase activity increases markedly asthe plants mature so that in old material cytochrome oxidaseactivity is only 10 per cent, of succinoxidase. 2. Disintegration of the mitochondria by vibration with ballotini,treatment with digitonin or incubation in the warm reveals anintra-mitochondrial cytochrome c oxidase probably active enoughto account for the fastest rates of succinate oxidation. 3. Succinic dehydrogenase activity is demonstrated and experimentswith p-chloromercuribenzoate indicate that it plays a part inthe oxidation of succinate. 4. Cyanide completely inhibits both external and internal cytochromeoxidase but even at the earliest stages it only reduces succinoxidaseby about 50 per cent. Antimycin A also inhibits succinoxidaseby about 50 per cent.     

Respiration Rate and Mitochondrial Oxidase Activity in Arum Spadix

1. Mitochondria prepared from young Arum spadix oxidise succinicand -ketoglutaric acids at about 300µl. O₂/hr./g. wetwt. but as the spadix ages the activity of the mitochondriaincreases more than tenfold. The increase results from a smallrise in the quantity of mitochondrial nitrogen brought downin the centrifuge and a larger rise in oxidase activity permg. mitochondrial nitrogen. 2. The rate of respiration of spadix slices has been measuredunder conditions in which it is not limited by slow diffusionof oxygen. Slices of young spadix respire at about 1,000–2,000µl. O₂/hr/g. but as the inflorescence opens the rate soarsto 20,000µl. O₂/hr/g. and the spadix becomes warm. 3. Cytochrome oxidase was assayed in mitochondria treated withdigitonin. There is at all times enough cytochrome oxidase toaccount for the rates of oxidation of succinate and -ketoglutarate;and enough to account for the relatively slow respiration ofthe young spadix slices but not for the fastest respirationof the mature spadix. 4. The respiration of spadix slices is found, in contradictionto an earlier report, to be sensitive to malonate.     

Characterization of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatases.

The catalytic properties of cuckoo-pint (Arum maculatum) mitochondrial adenosine triphosphatase have been analysed. The pH profile, effect of inhibitors, cold-stability and substrate specificity are characteristic of mitochondrial adenosine triphosphatases, although a high guanosine triphosphatase activity does appear to be restricted to plant mitochondrial adenosine triphosphatases. The kinetic properties of nucleoside 5'-triphosphate hydrolysis by membrane-bound and soluble enzymes have been studied by means of double-reciprocal plots. These plots were linear in the absence of an activating anion, which may indicate that the catalytic and/or regulatory mechanism of Arum maculatum adenosine triphosphatase is different from that of other enzyme preparations. It is suggested that the differences in subunit composition of plant and mammalian adenosine triphosphatases reported previously [Dunn, Slabas & Moore (1985) Biochem. J. 225, 821-824] are structurally, rather than functionally, significant.     

Partial Purification and Characterization of the Phosphate Transporter from Bovine Heart Mitochondria

A highly active phosphate transporter was extracted with octylglucoside from bovine heart submitochondrial particles that were first partially depleted of other membrane components. It was then partially purified by ammonium sulfate fractionation. After reconstitution of the transporter into liposomes prepared with a crude mixture of soybean phospholipids, the P_i/OH exchange, but not the P_i/P_i exchange, was stimulated three- to fourfold by valinomycin and nigericin in the presence of K⁺. Both P_i/OH and P_i/P_i exchange activities were sensitive to mercurials and other SH reagents. The rutamycin-sensitive ATPase complex from mitochondria was reconstituted together with the phosphate transporter and adenine nucleotide transporter into liposomes. After inhibition of externally located ATPase, the hydrolysis of ATP was sensitive to atractyloside and mersalyl.     

Alternative Oxidase Activity and the Ubiquinone Redox Level in Soybean Cotyledon and Arum Spadix Mitochondria during NADH and Succinate Oxidation

In Arum and soybean (Glycine max L.) mitochondria, the dependence of the alternative oxidase activity on the redox level of ubiquinone, with NADH and succinate as substrates, was studied, using a voltametric procedure to measure the ubiquinone redox poise in the mitochondrial membrane. The results showed that when the enzyme was activated by pyruvate the relationship between the alternative oxidase rate and the redox state of the ubiquinone pool was the same for both NADH and succinate oxidations. In the absence of pyruvate the alternative oxidase had an apparent lower affinity for ubiquinol. This was more marked with NADH than with succinate and was possibly due to pyruvate production during succinate oxidation or to an activation of the alternative oxidase by succinate itself. In Arum spadix (unlike soybean cotyledon) mitochondria, succinate oxidation via the alternative oxidase maintained the ubiquinone pool in a partially reduced state (60%), whereas NADH oxidation kept it almost completely reduced. Previous data comparing mitochondria from thermogenic and nonthermogenic tissues have not examined the full range of ubiquinone redox levels in both tissues, leading to the suggestion that the activity of alternative oxidase for Arum was different from nonthermogenic tissues. When the complete range of redox states of ubiquinone is used and the oxidase is fully activated, the alternative oxidase from thermogenic tissue (Arum) behaves similarly to that of nonthermogenic tissue (soybean).     

Partial Purification and Characterization of Polyphenol Oxidase Isozymes in Banana Bud

Polyphenol oxidase was extracted from banana buds in the presences of Triton X-100, isoascorbate, and Polyclar AT, and two isozymes I and II have been separated and partially purified by chromatographies on Butyl Toyopearl 650 and DEAE-cellulose. I and II had different mobility in polyacrylamide gel electrophoresis with optimum pHs of 6.8 and 5.5, respectively. Both enzymes showed the apparent K_m values of 0.5 mM for dopamine with substrate inhibitions at its higher concentrations. I and II were inhibited competitively by NaCI with the K_i values of 140 mM and 40 mM, respectively. I and II have a high heat stability, and 88 and 95% of the initial activities were retained after 1-hr incubation at 70°C, respectively.     

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.     

Isolation and Partial Characterization of a Cytochrome b Complex and Cytochrome Oxidase from Yeast Mitochondria

A cytochrome b complex and cytochrome oxidase have been purified 14- and 20-fold respectively from yeast submitochondrial particles by a simple procedure involving their spontaneous precipitation from a deoxycholate extract. The recovery of both proteins was almost quantitative. The specific heme contents were 11 and 8 nmoles/mg protein for the cytochrome b complex and cytochrome oxidase respectively and both were spectrally pure. Sodium dodecyl sulfate gel electrophoresis resolved the cytochrome b complex into seven distinct subunits with molecular weights 42, 000, 33, 000, 27, 500, 23, 000, 15, 500, 13, 000 and 10, 500. Cytochrome oxidase contained five bands with molecular weights 42, 000, 26, 500, 21, 000, 14, 000 and 10, 500. Much of the cytochrome b complex (and all of the cytochrome oxidase) could be resolubilized in aqueous buffer following precipitation from the deoxycholate extract. The fraction of the cytochrome b preparation which remained insoluble appeared identical to the soluble protein in terms of polypeptide composition but contained less phospholipid and bound detergent, suggesting that insolubility may result from interaction between hydrophobic regions otherwise occupied by amphiphiles. The soluble cytochrome b complex migrated as a single species upon analytical ultracentrifugation and column chromatography, and during electrophoresis on polyacrylamide gels. Triton X-100, urea, or bile salts, failed to dissociate the complex. These findings suggest that the subunits are tightly associated in situ.     

重组E．coli胆固醇氧化酶的分离纯化及其酶学性质研究

对重组E.coli产生的胆固醇氧化酶采用70%硫酸铵盐析、CM Sepharose FF离子层析、Phenyl Sepharose 6 Fast疏水层析、Sephadex G-75凝胶过滤,得到的胆固醇氧化酶在SDS-PAGE上呈单一蛋白质条带,酶的纯化倍数为93,收率为21%.部分酶学性质表明:酶的最适反应温度为37℃,最适反应pH7.5,热稳定范围在40℃以下,酶的pH稳定范围为6～9,分子量分别为50 kD和52 kD.酶动力学参数Km值及Vmax分别为8.2×10-5 mol/L和0.21 mmol/(L.min).     

Partial Purification and Characterization of Polyphenol Oxidase from Cocoyam, Xanthosoma sagittifolium

Polyphenol oxidase has been partially purified from Xanthosomasagittifolium. The enzyme showed activity towards pyrogallol,DL-ß-3,4-dihydroxyphenylalanine (DOPA) and catechol.Of these three, pyrogallol was the best substrate. The effectsof various compounds as inhibitors of the reaction catalysedby the enzyme were tested. p-Nitrophenol competitively inhibitedthe binding of both catechol and pyrogallol to the enzyme. Inhibitionby the substrate analogue, p-cresol was of the mixed type whilethiourea and diethyldithiocarbamate inhibited the enzyme uncompetitively.The approximate molecular weight of the enzyme determined bygel filtration was 47 000.     

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.     

Studying Lords and Ladies,Arum maculatum (L.)

Historical uses of Arum maculatum (L.) are summarized. Field and laboratory investigations show plants with purple and pale spadices are equally attractive to flies of the genus Psychoda; and these are attracted to the scent in the absence of visual stimuli. Over 75% of the flies which enter spikes escape. Temperature changes during spadix maturation, and spathe uncurling in the presence of flower tissue, indol-3-yl acetic acid, and gibberellic acid are investigated in the laboratory. Dilute solutions of both phytohormones cause significant spathe uncurling. Further investigations, suitable for project work in schools and colleges, are suggested.     

Pro-inflammatory effect of Arum maculatum lectin and role of resident cells

Arum maculatum agglutinin (AMA) is a monocot lectin isolated from tubers of Arum maculatum L. (Araceae) which exhibits different specificity towards oligo-mannosidic-type and N-acetyllactosaminic-type glycans. We have investigated the effect of this lectin on the cells of the immune system. Models of neutrophil migration in vivo, neutrophil chemotaxis in vitro and macrophage cultures were used to study the lectin inflammatory activity. When administered into rat peritoneal cavities, AMA (80, 200 and 500 microg/mL/cavity) induced significant and dose-dependent neutrophil migration. This effect was inhibited by incubation with alpha-methyl-d-mannoside. A 83% depletion in the number of resident cells following peritoneal lavage did not reduce the AMA-induced neutrophil migration, as compared to sham animals (not washed). However, pre-treatment with 3% thioglycolate which increases the peritoneal macrophage population by 236%, enhanced the neutrophil migration induced by AMA (200 microg/mL/cavity) (119%, p < 0.05). Reduction of peritoneal mast cell population by chronic treatment of cavities with compound 48/80 did not modify AMA-induced neutrophil migration. The neutrophil chemotaxy assay in vitro shows that the lectin (300 microg/mL) induces neutrophil chemotaxy (368% p < 0.05) compared to RPMI. Finally, injection into peritoneal cavities of supernatants from macrophage cultures obtained after stimulation with AMA (300 microg/mL) enhanced neutrophil migration (110% p < 0.05). Summarizing, our data suggest that A. maculatum agglutinin presents pro-inflammatory activity, inducing neutrophil migration by two ways, one which is independent on resident cells and another one dependent on the presence of these cells.     

The Development of Mitochondria in Arum Spadix

Electron micrographs of Arum spadix cells prepared at four stagesof development all showed abundant sections of mitochondriawith tubular ingrowths (microvilli). At the earliest stage (whenthe spadix cells were still dividing) there was an average of9 sections of microvilli per mitochondrion. In later stagesof development, when the cells were growing by elongation, thenumber of microvilli rose to 22. It was reported earlier that the succinoxidase activity of themitochondria of Arum spadix increased as the spadix developed,and it is now shown that the rise in enzyme activity parallelsthe increase in length of microvilli.     

Partial Purification and Characterization of Three NAD(P)H Dehydrogenases from Beta vulgaris Mitochondria

Mitochondria isolated from the taproot of beet (Beta vulgaris) were used in an effort to identify and partially purify the proteins constituting the exogenous NADH dehydrogenase. Three NAD(P)H dehydrogenases are released from these mitochondria by sonication, and these enzymes were partially purified using fast protein liquid chromatography. One of the enzymes, designated peak I, is capable of oxidizing NADPH and the β form of NADH. The other two activities, peaks II and III, oxidize only β-NADH. All three peaks are insensitive to divalent cation chelators and a complex I inhibitor, rotenone. The major component to peak I is a polypeptide with an apparent molecular mass of approximately 42 kilodaltons. Peak I activity was insensitive to platanetin, a specific inhibitor of the exogenous dehydrogenase, and insensitive to added Ca²⁺ or Mg²⁺. Peak I displayed a broad pH activity profile with an optimum between 7.5 and 8.0 for both NADPH and NADH. Purified peak II gave a single polypeptide of about 32 kilodaltons, had a pH optimum between 7.0 and 7.5, and was slightly stimulated by Ca²⁺ and Mg²⁺. As with peak I, platanetin had no effect on peak II activity. Peak III was not purified completely, but contained two major polypeptides with apparent molecular masses of 55 and 40 kilodaltons. This enzyme was not affected by Ca²⁺ and Mg²⁺, but was inhibited by platanetin. The peak III enzyme had a rather sharp pH optimum of approximately 6.5 to 6.6. The above data indicate that peak III activity is likely the exogenous NADH dehydrogenase.     

The Oxidative Phosphorylation and ATPase Activity of Arum spadix Mitochondria in Relation to Heat Generation

The respiration of Arum spadix mitochondria is coupled to asub-maximal stoichiometry of ATP synthesis. The P/O ratios associatedwith the oxidation of succinate or malate are decreased by antimycinand increased by m-chlorobenzhydroxamic acid, an inhibitor ofthe alternative oxidase. The mitochondrial ATPase activity of20–40 nmol (mg protein)^–1 min^–1 is independentof the maturity of the spadix and is unlikely to provide themechanism for heat production during the odoriferous stage,which probably results from an increase in the rate of electrontransport via the non-phosphorylating, cyanide-insensitive oxidase.