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R Douce

Publications and source records attributed to R Douce.

At least 181 records · Page 10Linked to original sources
PubMed1982

Isolation and characterization of metabolically competent mitochondria from spinach leaf protoplasts.

Intact mitochondria were prepared from spinach (Spinacia oleracea L. var. Kyoho) leaf protoplasts and purified by Percoll discontinuous gradient centrifugation. Assays of several marker enzymes showed that the final mitochondrial preparations obtained are nearly free from other contaminating organelles, e.g. chloroplasts, peroxisomes, and endoplasmic reticulum. These mitochondria oxidized malate, glycine, succinate, and NADH, tightly coupled to oxidative phosphorylation with high values of ADP to O ratio as well as respiratory control ratio. The rate of NADH oxidation was 331 nmoles O(2) per milligram mitochondrial protein per minute, which is comparable to that obtained by highly purified potato or mung bean mitochondria. However, the activity of glutamine synthetase was barely detectable in the isolated mitochondrial fraction. This finding rules out a hypothetical scheme (Jackson, Dench, Morris, Lui, Hall, Moore 1971 Biochem Soc Trans 7: 1122) dealing with the role of the mitochondrial glutamine synthetase in the reassimilation of NH(3), which is released during the step of photorespiratory glycine decarboxylation in green leaf tissues, but it is consistent with the photosynthetic nitrogen cycle (Keys, Bird, Cornelius, Lea, Wallsgrove, Miflin 1978 Nature (Lond) 275: 741), in which NH(3) reassimilation occurs outside the mitochondria.

Journal Article↗
PubMed1982

Polar lipid composition of a plastid ribosome-deficient barley mutant.

Green and white leaves of the barley mutant line ;albostrians' were compared for their polar lipid content and fatty acid composition. The mutant plastids of the white leaves have a double-layered envelope, but in contrast with the normal chloroplasts, lack 70 S ribosomes and thylakoids. In the green leaves, the amount of monogalactosyldiacylglycerol (MGDG) consistently exceeds the amount of digalactosyldiacylglycerol (DGDG) and the amount of galactolipids exceeds the amount of phospholipids. In contrast, in white leaves the amount of DGDG exceeds the amount of MGDG and the amount of phospholipids exceeds the amount of galactolipids. In white leaves, the galactolipid composition reflects the plastid envelope composition which is rich in DGDG, whereas in green leaves the galactolipid composition reflects the thylakoid composition which is rich in MGDG. These results demonstrate the likelihood that all the enzymes involved in galactolipid, sulfolipid and fatty acid synthesis are coded by the nuclear genome.

Journal Article↗
PubMed1982

A P Nuclear Magnetic Resonance Study of Intracellular pH of Plant Cells Cultivated in Liquid Medium.

(31)P nuclear magnetic resonance has been used to study the vacuolar and cytoplasmic pH of Acer pseudoplatanus, Catharanthus roseus, and Glycine max cells grown as cell suspensions. The adaptation of this technique to plant cells grown in liquid medium is described with emphasis on the removal of Mn(2+) and phosphate from the extracellular medium and on providing the O(2) supply of the cells in the nuclear magnetic resonance tube and the various problems of calibration. Aerobic and anaerobic cells show large differences in their glucose-6-phosphate, their cytoplasmic inorganic phosphate pools, and their cytoplasmic pH. Differences in the relative sizes of the cytoplasmic and vacuolar inorganic phosphate pools have been observed for the three cell strains studied.

Journal Article↗
PubMed1981

Localization of prenylquinones in the envelope of spinach chloroplasts.

The isolated and purified chloroplast envelope of spinach leaves contains, besides carotenoids, several prenylquinones as basic constituents: plastoquinone-9, phylloquinone K1, alpha-tocoquinone and the chromanol, alpha-tocopherol. The relative quinone and carotenoid composition of the envelope differs distinctively from that of the thylakoid membranes. The possible role of prenylquinones in metabolic envelope activities and the mediator function of the envelope in prenylquinone biosynthesis are discussed.

Chloroplasts↗
PubMed1981

Role of Glutamate-oxaloacetate Transaminase and Malate Dehydrogenase in the Regeneration of NAD for Glycine Oxidation by Spinach leaf Mitochondria.

During glycine oxidation by spinach leaf mitochondria, oxygen consumption showed a strong and transient inhibition upon addition of oxaloacetate or aspartate plus alpha-ketoglutarate. During the course of the inhibition, aspartate and alpha-ketoglutarate were stoichiometrically transformed into malate and glutamate.It is concluded that oxaloacetate formed by transamination is reduced by the malate dehydrogenase, which allows the regeneration of NAD(+) for glycine oxidation and, thus, by-passes the respiratory chain. Efficiency of a malate-glutamate/aspartate-alpha-ketoglutarate shuttle upon illumination and under in vivo conditions is discussed.

Journal Article↗
PubMed1981

Slow Passive Diffusion of Orthophosphate between Intact Isolated Chloroplasts and Suspending Medium.

Isolated spinach chloroplasts purified by isopycnic centrifugation in density gradients of Percoll were found to be highly intact, to be devoid of extrachloroplastic contaminations, and to retain a high rate of CO(2)-dependent O(2) evolution.When suspended in a medium which avoided rupture of the envelope, intact purified chloroplasts progressively lost their phosphate content by passive diffusion. This led to a slow decrease in the uptake of labeled 3-phosphoglyceric acid or orthophosphate (Pi) and in the rate of CO(2)-dependent O(2) evolution by isolated chloroplasts. Under these conditions, there was a good correlation between the rate of CO(2)-dependent O(2) evolution and the concentration of Pi in the stroma space. Addition of Pi to the suspending medium at a final concentration of 10 millimolar, which counterpoised the slow efflux of Pi from the chloroplasts, slowed considerably the decrease in the rate of CO(2)-dependent O(2) evolution.

Journal Article↗
PubMed1980

[Oxygen and temperature effects on the fatty acid composition in sycamore cells (Acer pseudoplatanus L.) (author's transl)].

Temperature and oxygen effects on the degree of unsaturation of membrane fatty acids have been investigated with sycamore cells in suspension culture. Sycamore cells were incubated with [14C]acetate at temperature varying from 15 to 25 degrees C and at O2 concentration from 12.5 to 305 muM. It was found that: (i) no significant difference was observed in the distribution of radioactivity between oleate and linoleate with different temperatures; (ii) in marked contrast, the aeration conditions during growth of plant cell cultures affected the fatty acid pattern of the total lipids: by maintaining the oxygen concentration below 60 muM, the molar proportion of oleate increased dramatically whereas that of the linoleate decreased. Under these conditions, the aeration of the culture medium (250 muM) induced a rapid transformation of oleate to linoleate. These results cast further doubt on the importance of the temperature on the degree of unsaturation of the fatty acids in sycamore cells, but confirmed evidence that the formation of unsurated fatty acids by plant cells was indeed controlled by the oxygen concentration in solution.

Cell Membrane Permeability↗
PubMed1980

Site of synthesis of geranylgeraniol derivatives in intact spinach chloroplasts.

Chloroplasts isolated from fully developed spinach leaves and incubated in the presence of isopentenyl pyrophosphate were able to synthesize rapidly geranylgeranyl chlorophyll alpha and geranylgeraniol. The biosynthesis of the geranylgeraniol derivatives from isopentenyl pyrophosphate is a compartimentalized process. The membrane fractions (thylakoid and envelope membranes) were essentially unable to synthesize geranylgeraniol, geranylgeranyl pyrophosphate and geranylgeranyl chlorophyll alpha. When stromal and thylakoid fractions were combined the capacity to synthesize geranylgeranyl chlorophyll alpha and geranylgeraniol was restored. When stromal and envelope membrane fractions were combined the capacity to synthesize gernylgeranyl pyrophosphate and geranylgeraniol was restored. The products of the reaction were discharged inside the lipid phase of the membranes.

Cell Compartmentation↗
PubMed1980

A precise localization of cardiolipin in plant cells.

Cardiolipin and cytochrome aa3 contents of isolated plant cells (sycamore cells) and their purified mitochondria were measured. Since the cardiolipin/cytochrome aa3 ratio was the same in the intact cells and in the isolated mitochondria it was strongly suggested that cardiolipin is present only in the mitochondria. Furthermore, outer and inner mitochondria membranes of purified sycamore cells and mung bean hypocotyl mitochondria were separated and it was shown that cardiolipin is localized in the inner mitochondrial membrane.

Cardiolipins↗
PubMed1980

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria.

Mitochondria isolated from spinach leaves oxidized malate by both a NAD+-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumuation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition. Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD+-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate. The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition. The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD+/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD+-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD+-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.

Bicarbonates↗
PubMed1980

Effect of NAD on Malate Oxidation in Intact Plant Mitochondria.

Potato tuber mitochondria oxidizing malate respond to NAD(+) addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD(+) in potato mitochondria, which prove to take up added NAD(+). This mechanism concentrates NAD(+) in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD(+)/NADH ratio.

Journal Article↗
PubMed1980

Distribution of radioactive lipids between envelopes and thylakoids from chloroplasts labelled in vivo.

Spinach leaves were labelled with 14CO2 for subsequent isolation of radioactive chloroplasts, which were separated into envelopes and thylakoids. The analyses carried out with the lipid extracts from these membranes were concerned with the following questions: are chloroplast envelopes a functional interface between endoplasmic reticulum and thylakoids, do they also play a predominant role in galactolipid biosynthesis in vivo, and is it possible to demonstrate galactolipid export from envelopes into thylakoids? Taken together the results show that lipid export is apparently too fast in vivo to be followed by the labelling and separation technique used, since thylakoid lipids contained always far more total label than envelope counterparts, whereas the specific activity of envelope lipids was higher. Phosphatidylcholine, which has been suggested to function as acyl carrier between endoplasmic reticulum and chloroplasts, was never found labelled to any extraordinary extent in envelopes. Envelopes may be regarded as small, but rapidly turned over lipid pools.

Chloroplasts↗
PubMed1980

Labelling in vivo and in vitro of molecular species of lipids from chloroplast envelopes and thylakoids.

Lipid mixtures from chloroplast envelope and thylakoid membrane were isolated after different labelling times in vivo and in vitro and separated into major components. The isolated compounds were subjected to analyses such as separation of molecular species, determination of radioactivity in fatty acids and water-soluble hydrolysis products and radio gas-liquid chromatography of fatty acid mixtures. In the case of monogalactosyl and digalactosyl diacylglycerol these procedures were also applied to several individual molecular species. To investigate the extent of de novo synthesis these species were also used for methylation studies and their fatty acids subjected to alpha-oxidation. In envelope membranes diacylglycerols and monogalactosyl diacylglycerols may each be separated into several distinct and non-mixing pools. Molecules made de novo with oligoene fatty acids are very efficient substrates for galactosylation in vivo. The time-dependent changes in patterns of galactolipid molecular species may indicate a desaturation of acyl chains operating in close contact to intact lipids. After isolation, envelopes incorporated UDP-[14C]galactose into completely different patterns of galactolipids and molecular species pointing to changed properties of this membrane system or to a loss of regulatory factors.

Chloroplasts↗
PubMed1979

Characterization of lipids from chloroplast envelopes.

The major neutral, glycolipids and phospholipids from envelopes of spinach chloroplasts were analyzed with respect to proportions, positional distribution and pairing of fatty acids. All specificities in the diacylglycerol portions of lipids known from previous analyses of lipids from whole leaves were also found in envelope lipids. Diacylglycerols and galactolipids share a common diacylglycerol portion. The only exception is digalactosyl diacylglycerol, which contains 18:3/16:0 but lacks 18:3/16:3 species reverting the distribution in other galactolipids. Phosphatidylcholine, phosphatidylglycerol and sulfoquinovosyl diacylglycerol are distinct from the galactolipids, because each one has a unique diacylglycerol profile. The diacylglycerol species present in phosphatidylcholine and galactolipids or free diacylglycerols do not provide evidence for a biogenetic relation between phosphatidylcholine and galactolipids at the level of envelopes.

Chloroplasts↗
PubMed1979

[Sulfate transport across the limiting double membrane or envelope, of spinach chloroplasts].

Evidence is presented for low rates of carrier-mediated uptake of sulphate, thiosulphate and sulphite into the stroma of the C3 plant Spinacia oleracea. Uptake of sulphate in the dark was followed using two techniques (1) uptake of sulphate [35S] as determined by silicon oil centrifugal filtration and (2) uptake as indicated by inhibition of CO2-dependent O2 evolution rates after addition of sulphate. Sulphate, thiosulphate and sulphite were transported across the envelope leading to an accumulation in the chloroplasts. Sulphate for each molecule of sulphate entering the chloroplast, one molecule of phosphate leaves the stroma, and vice-versa. The uptake of sulphate by isolated intact chloroplasts exchanging for internal free phosphate induced a lower rate of photophosphorylation, which in turn inhibited CO2-dependent O2 evolution. The presence, on the inner membrane of the chloroplasts envelope, of a specific sulphate carrier, distinct from the phosphate translocator, is discussed.

Adenosine Diphosphate↗
PubMed1978

Calcium-dependent lipolytic acyl-hydrolase activity in purified plant mitochondria.

Ageing of isolated potato mitochondria induced by CaCl2 resulted in rapid enzymatic hydrolysis of the membrane phospholipids with the liberation of free fatty acids. The enzyme responsible for this effect was identified as a membrane bound lipolytic acyl-hydrolase which was unmasked by CaCl2. The presence of this lipolytic acyl-hydrolase induced severe functional impairments in the mitochondrial oxidative and phosphorylative properties.

Adenosine Diphosphate↗