A low-molecular-weight ATPase from wheat-seedling mitochondria.

An ATPase which strikingly differed from the mitochondrial ATPases of yeast and of animal tissues was obtained when wheat seedling mitochondria, or electron transport particles derived from them, were subjected to ultrasonication and treated with ammonium sulphate. The enzyme which was purified by chromatography on Sephadex G-100 and DEAE-Sephadex (A50) failed to be inactivated as low as 43 000. The enzyme preparation was capable of hydrolysing ADP, in addition to ATP, and several other nucleoside diphosphates and triphosphates. In contrast to the ATPase of animal mitochondria, the activity of the wheat enzyme was almost as insensitive to oligomycin in intact mitochondria as it was after isolation from the organelles.     

Transfer of cytoplasmic organelles from an oligomycin-resistant Nicotiana cell suspension into tobacco protoplasts yielding oligomycin-resistant cybrid plants

Summary Oligomycin-resistant lines were derived from a Nicotiana sylvestris cell suspension, after N-nitroso-N-methylurea mutagenesis followed by selection in the presence of 0.4 g/ml oligomycin, a specific mitochondrial ATPase inhibitor. One of the lines, oli ^R38 was further analyzed to investigate the role of mitochondria in this resistance. The oli ^R38 line proved to be also highly resistant to venturicidin, another specific inhibitor of mitochondrial ATPase. By the donor-recipient protoplast-fusion procedure the cytoplasmic organelles of oli ^R38 were transferred to protoplasts of Line 92, a line of tobacco plants which contain the cytoplasmic organelles of N. undulata. Cell suspensions prepared from several cybrid plants, containing the cytoplasmic organelles of oli ^R38, exhibited the same level of oligomycin resistance as the oli ^R38 line.     

Molecular weight,amino acid composition and other properties of membrane-bound ATPase fromBacillus megaterium KM

The Ca²⁺-activated ATPase fromBacillus megaterium KM has a molecular weight of 379000 as determined by sedimentation equilibrium in the analytical ultracentrifuge and 410000 as determined from sedimentation and diffusion coefficients. These values compare closely with molecular weights estimated for similar ATPases fromStreptococcus faecalis and mitochondria. On polyacrylamide gel electrophoresis in sodium dodecylsulfate two classes of subunit of molecular weight 68000 and 65000 are seen. They seem to be present in approximately equal proportion. The amino acid analysis gives a minimum molecular weight of 6250 and the amino acid composition is extremely close to that ofS. faecalis. The enzyme is denatured at 55°C and insensitive to oligomycin or ruthenium red in either the membrane-bound or soluble forms. The ATPase is estimated to comprise approximately 1% of the total cytoplasmic membrane protein.     

Comparative studies of glyoxysomes from various Fatty seedlings

The separation of various organelles from cotton cotyledon (Gossypium hirsutum L.), cucumber cotyledon (Cucumis sativus L.), peanut cotyledon (Archis hypogaea L.), pine megagametophyte (Pinus ponderosa Laws), and watermelon cotyledon (Citrullus vulgaris Schrad.) by sucrose density gradient centrifugation was found to be similar to that described for castor bean endosperm (Ricinus communis L.). Equilibrium densities were 1.12 to 1.13 g cm³ for endoplasmic reticulum, 1.17 to 1.19 g/cm³ for mitochondria, and 1.25 g/cm³ for glyoxysomes. Isolated glyoxysomes from different fatty seedlings have striking similar specific activities of individual enzymes. The only exception is alkaline lipase activity which, when assayed with an artificial substrate, varies some 10-fold in glyoxysomes from different fatty seedlings. The properties of individual enzymes in glyoxysomes from different fatty seedlings are qualitatively similar as regard to sub-organelle localization and behavior in the presence of KCl and Triton X-100. In pine megagametophyte, the glyoxysomes and not the mitochondria are the intracellular site for the breakdown of stored lipid.     

Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation

A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers.     

Effects of centrifugal force and centrifugation time on the sedimentation of plant organelles

The effect of centrifugal force and length of centrifugation time on the sedimentation of plant organelles was determined for corn (Zea mays L.) root homogenates. A centrifugal force of 6000g for at least 20 minutes was necessary to pellet 90% of the mitochondrial marker (cytochrome c oxidase). This initial centrifugation step is optimal for separating mitochondria from microsomes, since cross-contamination of endoplasmic reticulum and plasma membrane vesicles with mitochondria is minimized. Centrifugal forces of 8000g or 10,000g for 20 minutes and 13,000g for 15 minutes pellet 90% of the mitochondrial marker; however, these centrifugation conditions also sediment more plasma membrane and endoplasmic reticulum.     

Isolation and properties of OSCP and an F1-ATPase binding protein from rat liver mitochondria - evidence against OSCP as the linking "stalk" between F1 and the membrane.

Oligomycin Sensitivity Conferral Protein (OSCP) and an F₁-ATPase Binding Protein were isolated from F₁-depleted rat liver mitochondrial membrane. Their molecular weights on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea were 22,500 and 8,500 respectively. When incubated with liver TUA (trypsin, urea and ammonia-treated) submitochondrial particles, the binding protein was effective in the binding of F₁ to the particles with the resultant particle-bound ATPase activity not oligomycin sensitive. When OSCP was then incubated with the reconstituted membrane-bound ATPase, its activity became oligomycin sensitive. These results suggest that, first; the binding protein, but not OSCP, connects F₁-ATPase to the membrane of rat liver mitochondria and maybe to the “stalk”, if indeed there is a stalk in mitochondrial membrane ATPase complex; and second; the function of OSCP is solely to render the ATPase activity sensitive to oligomycin and other similar inhibitors.     

Spegazzinine,a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I₅₀ was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca²⁺ and the proton ejection associated with the ATP-dependent Ca²⁺ uptake were not affected by the alkaloid.Oxidative phosphorylation and the P_i-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I₅₀, 50 μM) while the ATP-dependent reactions, reduction of NAD⁺ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I₅₀, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.     

The plant mitochondrial open reading frame orf221 encodes a membrane-bound protein

We have shown that the open reading frame orf221 is an active mitochondrial gene which encodes a novel mitochondrial polypeptide. The orf221 sequence is common to higher plants but absent in animal and fungal mitochondria. A mitochondrial polypeptide with an apparent molecular weight of 21 000 was detected with a polyclonal antibody raised against an ORF221 fusion protein. In organello translation followed by immunoprecipitation with the anti-ORF221 antibody demonstrated that this polypeptide is encoded by the orf221 gene in plant mitochondria. The ORF221 was found to be a mitochondrial membrane protein in normal (N), cms-T, and cms-C cytoplasms of several inbred lines of maize (Zea mays L.) and in other plant species.     

Monovalent ion stimulated adenosine triphosphatase from oat roots

Monovalent ion stimulated ATPase activity from oat (Avena sativa) roots has been found to be associated with various membrane fractions (cell wall, mitochondrial and microsomal) of oat roots. The ATPase requires Mg²⁺ (or Mn⁺²) but is further stimulated by K⁺ and other monovalent ions. The monovalent ions are ineffective in the absence of the divalent activating cation. The ATPase has been described with respect to monovalent ion specificity, temperature, pH, substrate specificity, and Mg²⁺ and K⁺ concentrations. It was further shown that oligomycin inhibits a part of the total ATPase activity and on the basis of the oligomycin sensitivity it appears that at least 2 membrane associated ATPases are being measured. The mitochondrial fraction is most sensitive to oligomycin and the microsomal fraction is least sensitive to oligomycin. The oligomycin insensitive ATPase appears to be stimulated more by K⁺ than the oligomycin sensitive ATPase.     

Identification, separation, and characterization of acyl-coenzyme A dehydrogenases involved in mitochondrial beta-oxidation in higher plants

The existence in higher plants of an additional β-oxidation system in mitochondria, besides the well-characterized peroxisomal system, is often considered controversial. Unequivocal demonstration of β-oxidation activity in mitochondria should rely on identification of the enzymes specific to mitochondrial β-oxidation. Acyl-coenzyme A dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus annuus L.) seeds, using as the enzyme assay the reduction of 2,6-dichlorophenolindophenol, with phenazine methosulfate as the intermediate electron carrier. Subcellular fractionation showed that this ACAD activity was associated with mitochondrial fractions. Comparison of ACAD activity in mitochondria and acyl-coenzyme A oxidase activity in peroxisomes showed differences of substrate specificities. Embryonic axes of sunflower seeds were used as starting material for the purification of ACADs. Two distinct ACADs, with medium-chain and long-chain substrate specificities, respectively, were separated by their chromatographic behavior, which was similar to that of mammalian ACADs. The characterization of these ACADs is discussed in relation to the identification of expressed sequenced tags corresponding to ACADs in cDNA sequence analysis projects and with the potential roles of mitochondrial β-oxidation in higher plants.     

Assessment of mitochondria as a compartment for phosphatidylinositol synthesis in Solanum tuberosum

The outer mitochondrial membrane is particularly rich in phosphatidylinositol (PtdIns), a phospholipid found in different amounts in all eukaryotic membranes, but not synthesized in situ by all. PtdIns is therefore subjected to traffic from the synthesizing membranes to the non-synthesizing ones. The contribution of mitochondria to the cell PtdIns pool has never been the focus of a specific study in plants, whereas in yeast, the presence of the enzyme responsible for synthesis, PtdIns synthase (PIS, cytidine 5′-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11), has clearly been demonstrated in mitochondria. As these organelles have now been shown to be responsible for the synthesis of several lipids, the present work aimed at evaluating mitochondria as a compartment for the synthesis of PtdIns in plants. The sub-cellular localization of PIS was studied in Solanum tuberosum L. by membrane fractionation, enzymatic analysis and by confocal microscopy in living cells. In potato, beside the endoplasmic reticulum, the activity of PIS was found to be tightly associated to mitochondria. Using a fluorescent reporter fusion, the enzyme was also found to be associated to these organelles. The enzyme was not present at the plasma membrane. A comparison of the localization in other cell systems suggests that the mitochondrial localization could be regulated.     

Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F₀ to F₁ ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N′-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme.     

Nitrogen transfer from forage legumes to nine neighbouring plants in a multi-species grassland

Legumes play a crucial role in nitrogen supply to grass-legume mixtures for ruminant fodder. To quantify N transfer from legumes to neighbouring plants in multi-species grasslands we established a grass-legume-herb mixture on a loamy-sandy site in Denmark. White clover (Trifolium repens L.), red clover (Trifolium pratense L.) and lucerne (Medicago sativa L.) were leaf-labelled with ¹⁵N enriched urea during one growing season. N transfer to grasses (Lolium perenne L. and xfestulolium), white clover, red clover, lucerne, birdsfoot trefoil (Lotus corniculatus L.), chicory (Cichorium intybus L.), plantain (Plantago lanceolata L.), salad burnet (Sanguisorba minor L.) and caraway (Carum carvi L.) was assessed. Neighbouring plants contained greater amounts of N derived from white clover (4.8?g?m^-2) compared with red clover (2.2?g?m^-2) and lucerne (1.1?g?m^-2). Grasses having fibrous roots received greater amounts of N from legumes than dicotyledonous plants which generally have taproots. Slurry application mainly increased N transfer from legumes to grasses. During the growing season the three legumes transferred approximately 40?kg?N ha^-1 to neighbouring plants. Below-ground N transfer from legumes to neighbouring plants differed among nitrogen donors and nitrogen receivers and may depend on root characteristics and regrowth strategies of plant species in the multi-species grassland.     

Blue light activates a specific protein kinase in higher plants

Blue light mediates the phosphorylation of a membrane protein in seedlings from several plant species. When crude microsomal membrane proteins from dark-grown pea (Pisum sativum L.), sunflower (Helianthus annuus L.), zucchini (Cucurbita pepo L.), Arabidopsis (Arabidopsis thaliana L.), or tomato (Lycopersicon esculentum L.) stem segments, or from maize (Zea mays L.), barley (Hordeum vulgare L.), oat (Avena sativa L.), wheat (Triticum aestivum L.), or sorghum (Sorghum bicolor L.) coleoptiles are illuminated and incubated in vitro with [γ-³²P]ATP, a protein of apparent molecular mass from 114 to 130 kD is rapidly phosphorylated. Hence, this system is probably ubiquitous in higher plants. Solubilized maize membranes exposed to blue light and added to unirradiated solubilized maize membranes show a higher level of phosphorylation of the light-affected protein than irradiated membrane proteins alone, suggesting that an unirradiated substrate is phosphorylated by a light-activated kinase. This finding is further demonstrated with membrane proteins from two different species, where the phosphorylated proteins are of different sizes and, hence, unambiguously distinguishable on gel electrophoresis. When solubilized membrane proteins from one species are irradiated and added to unirradiated membrane proteins from another species, the unirradiated protein becomes phosphorylated. These experiments indicate that the irradiated fraction can store the light signal for subsequent phosphorylation in the dark. They also support the hypothesis that light activates a specific kinase and that the systems share a close functional homology among different higher plants.     

Diversity and reductive evolution of mitochondria among microbial eukaryotes

All extant eukaryotes are now considered to possess mitochondria in one form or another. Many parasites or anaerobic protists have highly reduced versions of mitochondria, which have generally lost their genome and the capacity to generate ATP through oxidative phosphorylation. These organelles have been called hydrogenosomes, when they make hydrogen, or remnant mitochondria or mitosomes when their functions were cryptic. More recently, organelles with features blurring the distinction between mitochondria, hydrogenosomes and mitosomes have been identified. These organelles have retained a mitochondrial genome and include the mitochondrial-like organelle of Blastocystis and the hydrogenosome of the anaerobic ciliate Nyctotherus. Studying eukaryotic diversity from the perspective of their mitochondrial variants has yielded important insights into eukaryote molecular cell biology and evolution. These investigations are contributing to understanding the essential functions of mitochondria, defined in the broadest sense, and the limits to which reductive evolution can proceed while maintaining a viable organelle.     

Mitochondrial Activity and Ethanol Accumulation in Ice-encased Winter Cereal Seedlings

Cold-hardened dark-grown seedlings of winter wheat (Triticum aestivum L.) and winter rye (Secale cereale L.) are killed during total encasement in ice at −1 C at a rate related to the initial cold hardiness of the cultivars. Few plants remain alive after 7 days of encasement. Nonhardened seedlings are rapidly killed in ice. The respiratory properties of mitochondria isolated from plants after increasing periods of ice encasement decline slowly, and activity is little impaired when intact plants are about 50% killed. Electron microscopy indicates that mitochondrial structure is not disrupted until 3 weeks of ice encasement. Ethanol accumulates in hardened and nonhardened plants in ice, but at levels which are not toxic to the plants.     

Response of mitochondrial ATPase activity to uncouplers in isolated organelles and whole cells of Zajdela hepatoma

ATPase activity of coupled Zajdela hepatoma mitochondria was rendered uncoupler-sensitive by decreasing free fatty acids content in mitochondria or by preincubation of mitochondria with ATP prior to the addition of an uncoupler. The latter treatment resulted in an accelerated transport of ATP into the organelles. The effect of carbonylcyanide-m-chlorophenylhydrazone and oligomycin on the decrease of the ATP content in whole Zajdela hepatoma cells indicated that the hepatoma mitochondrial ATPase is stimulated by uncouplers $\text{in}\text{vivo}$. The conclusion is that the uncoupler-insensitive ATPase activity of coupled Zajdela hepatoma mitochondria is exhibited only by isolated organelles and results from a reduced $\text{ATP}\text{ADP}$ translocase activity.     

Subcellular Distribution of O-Acetylserine(thiol)lyase in Cauliflower (Brassica oleracea L.) Inflorescence

The subcellular localization of O-acetyiserine(thiol)lyase (EC 4.2.99.8) in nongreen tissue from higher plants has been studied using purified proplastids, mitochondria, and protoplasts from cauliflower (Brassica oleracea L.) buds as a source of subcellular fractions. O-Acetylserine(thiol)lyase has been detected in both organelles (proplastids and mitochondria) and a cytosolic extract obtained by protoplast fractionation. We confirmed these observations, demonstrating that a form of the enzyme different in global charge and separated from others by anion-exchange chromatography corresponded to each subcellular location. Our observations are consistent with the need for cysteine biosynthesis in each subcellular compartment where the synthesis of proteins occurs.