Search PubMed⌕ Search

Biomedical subjects

J Joyard

Publications and source records attributed to J Joyard.

At least 37 records · Page 2Linked to original sources
PubMed1994

Localization of ferrochelatase activity within mature pea chloroplasts.

Precise localization within the chloroplast of the membrane-bound enzymes involved in heme and chlorophyll biosynthetic pathways is of central importance for better understanding the regulation of the carbon flow into these two pathways. In this study we examine the localization of ferrochelatase activity within mature pea chloroplasts. Our results provide evidence that chloroplast ferrochelatase is associated only with thylakoid membranes. The presence of ferrochelatase in chloroplast thylakoids emphasizes the role of this membrane system in chloroplast protoheme biosynthesis. Furthermore, these results raise the possibility that heme and chlorophyll biosynthesis are compartmentalized in two distinct membrane systems within mature chloroplasts.

Chloroplasts↗
PubMed1994

Kinetic properties of monogalactosyldiacylglycerol synthase from spinach chloroplast envelope membranes.

We have investigated the functioning of monogalactosyldiacylglycerol (MGDG) synthase activity partially purified from spinach chloroplast envelope membranes, using mixed micelles containing diacylglycerol (the substrate for MGDG synthase), CHAPS (3-[(cholamidopropyl)dimethylammonio]-1- propanesulfonic acid), and phosphatidylglycerol. The presence of this anionic phospholipid was essential for optimal MGDG synthase activity because it strongly improves diacylglycerol solubilization by CHAPS. We have demonstrated that the "surface dilution" kinetic model proposed by Deems et al. (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) is valid for MGDG synthase assayed in mixed micelles within a narrow range of CHAPS concentration. However, the experimental conditions we have set up in this study led to the description of defined equilibrium and kinetic parameters of the interaction of the envelope MGDG synthase with diacylglycerol. Two-substrate kinetic studies were performed with varied UDP-galactose molar concentrations and varied dioleoylglycerol surface concentrations. The families of reciprocal plots obtained were shown to intersect at a single point of the 1/[substrate] axis thus demonstrating that MGDG synthase is a sequential, either random or ordered, bireactant system. Therefore, MGDG synthase possesses two distinct and independent substrate-binding sites, a hydrophilic one for UDP-galactose and a hydrophobic one for diacylglycerol. The dependence of kinetic parameters on the diacylglycerol mol fraction allows a comparison of the affinity of the enzyme for a wide range of diacylglycerol molecular species. The Km values obtained were ranging between 0.0089 mol fraction (52 microM) for dilinoleoylglycerol (18:2/18:2) to 0.0666 mol fraction (416 microM) for distearoylglycerol (18:0/18:0), but the differences observed were not really related to the unsaturation of the molecule since the Km value for dilinoleoylglycerol was much lower than that (0.040 mol fraction) for dilinoleoylglycerol (18:3/18:3). The Km values for dioleoylglycerol (18:1/18:1) and for the diacylglycerol molecular species synthesized within chloroplasts, i.e. containing 18:1/16:0, were in the average range, i.e. lower than 0.030 mol fraction (around 170 microM).

Chloroplasts↗
PubMed1993

Detection and characterization of a plastid envelope DNA-binding protein which may anchor plastid nucleoids.

Chloroplast DNA (cpDNA) binds to the envelope membrane of actively dividing chloroplasts (plastids) in young pea leaves. South-western blotting was used to identify and characterize the protein involved in the binding of cpDNA to the envelope membrane. A 130 kDa protein in the inner chloroplast (plastid) envelope membrane binds specific sequences within the cpDNA. These included a 0.41 kbp sequence located upstream of the psaAB gene, a 0.57 kbp sequence located downstream of the petA gene and a 1.2 kbp sequence located within the rpoC2 gene. The protein was detected in the envelope membrane of young pea leaves in which the cpDNA had been located by fluorescence microscopy at the chloroplast periphery, whereas it was undetectable in mature leaves. We therefore propose that the 130 kDa protein is involved in the binding of cpDNA to the envelope membrane, and named it plastid envelope DNA-binding protein.

Amino Acid Sequence↗
PubMed1992

Feedback inhibition of phosphatidate phosphatase from spinach chloroplast envelope membranes by diacylglycerol.

Because the envelope phosphatidate phosphatase plays a pivotal role in chloroplast glycerolipid metabolism, we have analyzed whether diacylglycerol could be a regulatory factor of the enzyme. Using isolated envelope membranes in which the level of diacylglycerol was modified by thermolysin treatment of intact chloroplasts to destroy the galactolipid:galactolipid galactosyltransferase, we have demonstrated that phosphatidate phosphatase activity was reduced when the membrane was enriched in diacylglycerol. All 1,2-diacylglycerol molecular species assayed were demonstrated to inhibit the enzyme to about the same extent. Kinetic studies with envelope from thermolysin-treated chloroplasts were performed in the absence and presence of diacylglycerol, and diacylglycerol was shown to be a powerful competitive inhibitor of the reaction. Finally, using isolated intact spinach chloroplasts, we have demonstrated that in situ phosphatidate phosphatase activity can be modulated by the level of diacylglycerol present in the membrane. The relevance of phosphatidate phosphatase inhibition by diacylglycerol in the regulation of chloroplast glycerolipid biosynthesis is discussed.

Cell Fractionation↗
PubMed1992

Localization within chloroplasts of protoporphyrinogen oxidase, the target enzyme for diphenylether-like herbicides.

Plant protoporphyrinogen oxidase is of particular interest since it is the last enzyme of the common branch for chlorophyll and heme biosynthetic pathways. In addition, it is the target enzyme for diphenyl ether-type herbicides, such as acifluorfen. Two distinct methods were used to investigate the localization of this enzyme within Percoll-purified spinach chloroplasts. We first assayed the enzymatic activity by spectrofluorimetry and we analyzed the specific binding of the herbicide acifluorfen, using highly purified chloroplast fractions. The results obtained give clear evidence that chloroplast protoporphyrinogen oxidase activity is membrane-bound and is associated with both chloroplast membranes, i.e. envelope and thylakoids. Protoporphyrinogen oxidase specific activity was 7-8 times higher in envelope membranes than in thylakoids, in good agreement with the number of [3H]acifluorfen binding sites in each membrane system: 21 and 3 pmol/mg protein, respectively, in envelope membranes and thylakoids. On a total activity basis, 25% of protoporphyrinogen oxidase activity were associated with envelope membranes. The presence of protoporphyrinogen oxidase in chloroplast envelope membranes provides further evidence for a role of this membrane system in chlorophyll biosynthesis. In contrast, the physiological significance of the enzyme associated with thylakoids is still unknown, but it is possible that thylakoid protoporphyrinogen oxidase could be involved in heme biosynthesis.

Chloroplasts↗
PubMed1991

Purification and characterization of E37, a major chloroplast envelope protein.

We have purified to homogeneity E37, the second major polypeptide of the inner membrane of the chloroplast envelope. The protein was retained on a Mono S column at pH 7, indicating it is a basic protein. After cyanogen cleavage, the protein was partially sequenced at 2 different sites. The sequence is compared with the deduced amino acid sequence of a cDNA coding for a 37 kDa envelope polypeptide recently published by Dreses-Werringloer et al. (Eur. J. Biochem. (1991) 195, 361-368.)

Amino Acid Sequence↗
PubMed1990

Envelope membranes from mature spinach chloroplasts contain a NADPH:protochlorophyllide reductase on the cytosolic side of the outer membrane.

Using fluorescence spectroscopy, we have demonstrated that isolated envelope membranes from mature spinach chloroplasts catalyze the phototransformation of endogenous protochlorophyllide into chlorophyllide in presence of NADPH, but not in presence of NADH. Protochlorophyllide reductase was characterized further using monospecific antibodies (anti-protochlorophyllide reductase) raised against the purified enzyme from oat. In mature spinach chloroplasts, protochlorophyllide reductase is present only in envelope membranes. We have demonstrated that the envelope protochlorophyllide reductase, a 37,000-dalton polypeptide, is only a minor envelope component and is present on the outer surface of the outer envelope membrane. This conclusion is supported by several lines of evidence: (a) the envelope polypeptide that was immunodecorated with anti-protochlorophyllide reductase can be distinguished from the major 37,000-dalton envelope polypeptide E37 (which was identified by monospecific antibodies) only after two-dimensional polyacrylamide gel electrophoresis; (b) the envelope protochlorophyllide reductase was hydrolyzed when isolated intact chloroplasts were incubated in presence of thermolysin; and (c) isolated intact chloroplasts strongly agglutinate when incubated in presence of antibodies raised against protochlorophyllide reductase. These results demonstrate that major differences exist between chloroplasts and etioplasts with respect to protochlorophyllide reductase levels and localization. The presence on the chloroplast envelope membrane of both the substrate (protochlorophyllide) and the enzyme (protochlorophyllide reductase) necessary for chlorophyllide synthesis could have major implications for the understanding of chlorophyll biosynthesis in mature chloroplasts.

Chloroplasts↗
PubMed1990

Intraorganelle localization and substrate specificities of the mitochondrial acyl-CoA: sn-glycerol-3-phosphate O-acyltransferase and acyl-CoA: 1-acyl-sn-glycerol-3-phosphate O-acyltransferase from potato tubers and pea leaves.

The mitochondrial sn-glycerol-3-phosphate and 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from potato tubers and pea leaves were investigated with respect to their intraorganelle localization, their positional and substrate specificities, and their fatty acid selectivities. In mitochondria from potato tubers both enzymes were found to be located in the outer membrane. The 1-acyl-sn-glycerol-3-phosphate O-acyltransferase of pea mitochondria showed the same intraorganelle localization whereas the sn-glycerol-3-phosphate O-acyltransferase behaved like a soluble protein of the intermembrane space. The sn-glycerol-3-phosphate O-acyltransferase of both potato and pea mitochondria used sn-glycerol-3-phosphate but not dihydroxyacetone phosphate as acyl acceptor and exclusively catalyzed the formation of 1-acyl-sn-glycerol-3-phosphate which subsequently served as substrate for the second acylation reaction at its C-2 position. Both acyltransferases of potato as well as pea mitochondria showed higher activities with acyl-CoA than with the corresponding acyl-(acyl carrier protein) thioesters. When different acyl-CoA thioesters were offered separately, the sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed no fatty acid specificity whereas the enzyme of pea mitochondria revealed one for saturated acyl groups. On the other hand, the mitochondrial 1-acyl-sn-glycerol-3-phosphate O-acyltransferases from both potato tubers and pea leaves were more active on unsaturated than on saturated acyl-CoA thioesters. Furthermore, these enzymes preferentially used oleoyl- and linoleoyl-CoA when they were offered in a mixture with saturated ones, although the fatty acid selectivity of the pea enzyme was less pronounced than that of the potato enzyme. The sn-glycerol-3-phosphate O-acyltransferase of potato mitochondria displayed a slight preference for saturated acyl groups.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed1990

Do thylakoids really contain phosphatidylcholine?

Isolated intact spinach chloroplasts were incubated with phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) under mild experimental conditions in which only the phosphatidylcholine localized in the cytosolic leaflet of the outer envelope membrane can be hydrolyzed. Thylakoids, which were protected from phospholipase C degradation, were subsequently prepared from the phospholipase C-treated chloroplasts and found to be devoid of phosphatidylcholine. Previously reported occurrences of phosphatidylcholine in thylakoid preparations probably reflect contamination of the thylakoids by envelope membranes. In the present work, contamination of thylakoids by envelope membranes was determined by measuring the 1,2-diacylglycerol 3-beta-galactosyltransferase [monogalactosyldiacylglycerol (MGDG) synthase; UDPgalactose: 1,2-diacylglycerol 3-beta-D-galactosyltransferase, EC 2.4.1.46] in the different chloroplast subfractions. We conclude that phosphatidylcholine is not present in highly purified thylakoids. Phosphatidylcholine is also absent from prokaryotic cyanobacterial membranes, and our results are in agreement with the endosymbiotic origin of higher plant chloroplasts.

Journal Article↗
PubMed1989

Purification and characterization of acetohydroxyacid reductoisomerase from spinach chloroplasts.

Acetohydroxyacid reductoisomerase was purified over 400-fold to a specific activity of 62 nkat.mg-1, with 2-aceto-2-hydroxybutyrate as substrate, from the stroma of spinach leaf chloroplasts. The enzyme was not intrinsically membrane bound. The native enzyme was a tetramer with a subunit Mr of 59,000. The activity was optimum between pH 7.5 and 8.5. The apparent Km for 2-acetolactate was 25 microM and for 2-aceto-2-hydroxybutyrate was 37 microM. The enzyme required Mg2+ and the Vmax. was attained at physiological Mg2+ concentrations. NADP+ competitively inhibited the reaction when NADPH was the varied substrate. The native enzyme eluted from Mono-Q ion-exchange resins as three distinct peaks of activity. This elution pattern was preserved when the peaks were combined, dialysed and re-chromatographed. Each form exhibited identical Mr of 59,000 after SDS/polyacrylamide gel electrophoresis (PAGE), whereas they were easily distinguishable from each other after PAGE under non-denaturing conditions. These results provide evidence for the existence of multiple forms of acetohydroxyacid reductoisomerase in chloroplasts isolated from spinach leaves.

2-Acetolactate Mutase↗
PubMed1989

Effect of sulphate on glutamate synthesis by intact spinach (Spinacia oleracea) chloroplasts.

During the course of NH4+ (or NO2-)-plus-alpha-oxoglutarate-dependent O2 evolution in spinach (Spinacia oleracea) chloroplasts, glutamate was continuously excreted out of the chloroplasts. Under these conditions, for each molecule of NO2- or NH4+ which disappeared, one molecule of glutamate accumulated in the medium and the concentration of glutamate in the stroma space was maintained constant. SO4(2-) (or SO3(2-) behave as inhibitors of NH4+ incorporation into glutamate by intact chloroplasts. This considerable inhibition of glutamate synthesis by SO4(2-) was correlated with a rapid decline in the stromal Pi concentration. The reloading of stromal Pi with either external Pi or PPi4- relieved SO4(2-)-induced inhibition of glutamate synthesis by intact chloroplasts. It was concluded that SO4(2-) induced a rapid efflux of stromal Pi out of the chloroplast, leading to a limitation of ATP synthesis and therefore to an arrest of ATP-dependent glutamine synthetase functioning.

Brassica↗
PubMed1989

Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds.

The availability of methods to fractionate non-green plastids and to prepare their limiting envelope membranes [Alban, Joyard & Douce (1988) Plant Physiol. 88, 709-717] allowed a detailed analysis of the biosynthesis of lysophosphatidic acid, phosphatidic acid, diacylglycerol and monogalactosyl-diacylglycerol (MGDG) in two different types of non-green starch-containing plastids: plastids isolated from cauliflower buds and amyloplasts isolated from sycamore cells. An enzyme [acyl-ACP (acyl carrier protein):sn-glycerol 3-phosphate acyltransferase) recovered in the soluble fraction of non-green plastids transfers oleic acid from oleoyl-ACP to the sn-1 position of sn-glycerol 3-phosphate to form lysophosphatidic acid. Then a membrane-bound enzyme (acyl-ACP:monoacyl-sn-glycerol 3-phosphate acyltransferase), localized in the envelope membrane, catalyses the acylation of the available sn-2 position of 1-oleoyl-sn-glycerol 3-phosphate by palmitic acid from palmitoyl-ACP. Therefore both the soluble phase and the envelope membranes are necessary for acylation of sn-glycerol 3-phosphate. The major difference between cauliflower (Brassica oleracea) and sycamore (Acer pseudoplatanus) membranes is the very low level of phosphatidate phosphatase activity in sycamore envelope membrane. Therefore, very little diacylglycerol is available for MGDG synthesis in sycamore, compared with cauliflower. These findings are consistent with the similarities and differences described in lipid metabolism of mature chloroplasts from 'C18:3' and 'C16:3' plants (those with MGDG containing C18:3 and C16:3 fatty acids). Sycamore contains only C18 fatty acids in MGDG, and the envelope membranes from sycamore amyloplasts have a low phosphatidate phosphatase activity and therefore the enzymes of the Kornberg-Pricer pathway have a low efficiency of incorporation of sn-glycerol 3-phosphate into MGDG. By contrast, cauliflower contains MGDG with C16:3 fatty acid, and the incorporation of sn-glycerol 3-phosphate into MGDG by the enzymes associated with envelope membranes is not limited by the phosphatidate phosphatase. These results demonstrate that: (1) non-green plastids employ the same biosynthetic pathway as that previously established for chloroplasts (the formation of glycerolipids is a general property of all plastids, chloroplasts as well as non-green plastids), (2) the envelope membranes are the major structure responsible for the biosynthesis of phosphatidic acid, diacylglycerol and MGDG, and (3) the enzymes of the envelope Kornberg-Pricer pathway have the same properties in non-green starch-containing plastids as in mature chloroplasts from C16:3 and C18:3 plants.

Acyltransferases↗
PubMed1988

Lipid requirement and kinetic studies of solubilized UDP-galactose:diacylglycerol galactosyltransferase activity from spinach chloroplast envelope membranes.

We have demonstrated a lipid requirement for the UDPgalactose:1,2-diacylglycerol 3-beta-D-galactosyl-transferase (or monogalactosyldiacylglycerol synthase; EC 2.4.1.46), an enzyme involved in the biosynthesis of monogalactosyldiacylglycerol, solubilized from chloroplast envelope membranes and partially purified by hydroxyapatite chromatography. The enzyme fraction was highly delipidated (<0.1 mg of lipid per mg of protein), and addition of lipids extracted from chloroplast membranes was necessary to reveal the activity. Acidic glycerolipids, and especially phosphatidylglycerol, were the best activators of the enzyme. The preparation of a delipidated enzyme fraction and the development of optimal assay conditions were prerequisites for the determination of the kinetic parameters for the hydrophobic substrate of the enzyme, diacylglycerol. In addition, we have demonstrated the existence of two substrate-binding sites: a hydrophobic one for diacylglycerol and a hydrophilic one for UDP-galactose.

Journal Article↗
PubMed1988

Preparation and characterization of envelope membranes from nongreen plastids.

We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes.

Journal Article↗
PubMed1988

Characterization of elemental sulfur in isolated intact spinach chloroplasts.

Incubation of intact spinach (Spinacia oleracea L.) chloroplasts in the presence of (35)SO(4) (2-) resulted in the light-dependent formation of a chloroform-soluble sulfur-containing compound distinct from sulfolipid. We have identified this compound as the most stable form (S(8)) of elemental sulfur (S(0), valence state for S = O) by mass spectrometry. It is possible that elemental sulfur (S(0)) was formed by oxidation of bound sulfide, i.e. after the photoreduction of sulfate to sulfide by intact chloroplasts, and released as S(8) under the experimental conditions used for analysis.

Journal Article↗