Prenyl lipid and fatty-acid synthesis in isolatedAcetabularia chloroplasts

A gentle procedure allowed the isolation of intact and highly active chloroplasts from the unicellular green algaAcetabularia mediterranea. These chloroplasts incorporated carbon from NaH¹⁴CO₃ into fatty acids and prenyl lipids at a rate of about 20–50 nmol carbon· (mg chlorophyll)⁻¹·h⁻¹. Most of the fatty acids formed in vitro were esterified in galactolipids. The main prenyl lipids synthesized were the chlorophyll side chain, intermediates of the carotenogenic path, α-and β-carotene, as well as lutein. Large amounts of [1-¹⁴C]acetate were incorporated, but exclusively into fatty acids.Isopentenyl diphosphate was a good substrate for prenyl-lipid formation in hypotonically treated chloroplasts. The envelope of intact chloroplasts, however, was impermeable to this compound. Intermediates of the mevalonate pathway were not accepted as precursors under conditions whereisopentenyl diphosphate was well incorporated. The results show that the lipid biosynthetic pathways in the plastids ofAcetabularia, a member of the ancient family of Dasycladaceae, are very similar to those in higher-plant plastids. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Synthesis of acyl-CoAs by isolated spinach chloroplasts in relation to added CoA and ATP

The kinetics of incorporation of [2-¹⁴C] acetate into lipids and acyl-CoAs in relation to added CoA and ATP by isolated spinach chloroplasts have been examined. The effect of the concentration of these cofactors on lipid and acyl-CoA synthesis was also studied. In the absence of cofactors, or when only one was present, the incorporation was very low and went mainly into lipids. When both cofactors were present a strong stimulation of both activities occurred. After 25 min, acyl-CoAs were more strongly labeled than lipids and both activities continued linearly for at least 60 min.Abbreviations ACP acyl carrier protein - FFA free fatty acids     

Prenyl lipid formation in spinach chloroplasts and in a cell-free system of Synechococcus (Cyanobacteria): polyprenols,chlorophylls, and fatty acid prenyl esters

Isolated chloroplasts from spinach leaf cells, chloroplast subfractions, and a cell-free system of the cyanobacterium Synechococcus CCAP 6312 incorporated [1-¹⁴C]isopentenyl pyrophosphate in high yields into prenyl lipids. Products were polyprenols (C₂₀, C₄₅) chlorophylls, quinoid compounds, and fatty acid prenyl esters; prenyl pyrophosphates occurred in trace amounts, and carotenes were only formed to a limited extent in the Synechococcus system. The formation of fatty acid prenyl esters, which is described here for the first time, was found to occur in two different ways in the chloroplast system; by an acyl-CoA: polyprenol acyltransferase reaction associated with the envelope membranes and by a transesterification reaction from chlorophyll associated with the thylakoids. Endogenous fatty acid prenyl esters made up about 3% by weight of total lipids in spinach chloroplasts and were also found to be natural constituents of the cyanobacterial cells.Abbreviations Chl chlorophyll - Chl_GG chlorophyll a containing a geranylgeranyl side chain - IPP isopentenyl pyrophosphate     

The action of phosphatidate phosphatase on the fatty-acid composition of safflower triacylglycerol and spinach glycerolipids

The microsomal phosphatidate phosphatase (EC 3.1.3.4) in maturing seeds of safflower (Carthamus tinctorius L.) was specific and selective for unsaturated phosphatidates. The relative order of specificity for phosphatidate molecular species was 1,2-dilinoleoyl = 1,2-dioleoyl > 1-palmitoyl-2-oleoyl > 1,2-dilauroyl = 1,2-dimyristoyl > 1,2-dipalmitoyl. The order of selectivity was similar to that of the specificity. The broad selectivity for unsaturated phosphatidate species (1,2-di-unsaturated-acyl and 1-saturated-acyl-2-unsaturatedacyl) led us to conclude that the phosphatidate-phosphatase reaction does not, or only very little, affect the fatty-acid composition of the diacylglycerol product and in turn the fatty-acid composition of triacylglycerol in safflower oil. As compared with the safflower microsomal enzyme, the chloroplast phosphatidate phosphatase of spinach (Spinacia oleracea L.) leaves showed a broader specificity. This agreed with the selectivity profile indicated by labelling patterns of phosphatidate and diacylglycerol synthesized from [¹⁴C]acetate in spinach chloroplasts (S.E. Gardiner et al. 1984, Biochem. J. 224, 637–643).Abbreviations BSA bovine serum albumin - FA fatty acid I thank Nippon Oil & Fats Co., Amagasaki, Japan, for providing pure oleic acid.     

Comparison of the kinetic properties,inhibition and labelling of the phosphate translocators from maize and spinach mesophyll chloroplasts

The kinetic properties of the phosphate translocator from maize (Zea mays L.) mesophyll chloroplasts have been determined. We have used a double silicone-oil-layer centrifugation system in order to obtain true initial uptake rates in forward-reaction experiments. In addition, it was possible to perform back-exchange experiments and to study the effects of illumination and of preloading the chloroplasts with different substrates on transport. It is shown that the phosphate translocator from mesophyll chloroplasts of maize, a C₄ plant, transports inorganic phosphate and phosphorylated C₃ compounds in which the phosphate group is linked to the C₃ atom (e.g. 3-phosphoglycerate and triose phosphate). The affinities of the transported metabolites towards the translocator protein are about one order of magnitude higher than in mesophyll chloroplasts from the C₃ plant, spinach. In contrast to the phosphate translocator from C₃-mesophyll chloroplasts, that of C₄-mesophyll chloroplasts catalyzes in addition the transport of C₃ compounds where the phosphate group is attached to the C₂ atom (e.g. 2-phosphoglycerate, phosphoenolpyruvate). The phosphate translocator from both chloroplast types is strongly inhibited by pyridoxal-5-phosphate (PLP), 2,4,6-trinitrobenzenesulfonic acid and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). In the case of the spinach translocator protein these inhibitors were shown to react with the same amino-acid residue at the substrate binding site, and one molecule of either DIDS or PLP is obviously required per substrate binding site for the inactivation of the translocation process. In the functionally active dimeric translocator protein only one substrate-binding site appears to be accessible at a particular time, indicating that the site might be exposed to each side of the membrane in turn. Using [³H]-H₂DIDS for the labelling of maize mesophyll envelopes the radioactivity was found to be associated with two polypeptides of about 29 and 30 kDa. Since Western-blot analysis showed that only the 30 kDa polypeptide reacted with an antiserum directed against the spinach phosphate translocator protein it is suggested that this polypeptide presumably represents the phosphate translocator from maize mesophyll chloroplasts.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - PEP phosphoenolpyruvate - 2-,3-PGA 2-,3-phosphoglycerate - PLP pyridoxal-5-phosphate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TNBS 2,4,6-trinitrobenzenesulfonic acid - triose P triose phosphate This work was supported by the Deutsche Forschungsgemeinschaft     

Regulation of adenylate levels in intact spinach chloroplasts

Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per min per mg protein was found, whereas a lower value (6 nmol per min per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per min per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per min per mg protein) and contained two of these three phosphorylase forms.Abbreviations G1P Glucose 1-phosphate - P_i orthophosphate - Tris Tris (hydroxymethyl)aminomethane - MES 2(N-morpholino)ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Purification and characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts

Thylakoid-bound superoxide dismutase (SOD; EC 1.15.1.1) was solubilized by Triton X-100 from spinach and purified to a homogeneous state. The molecular weight of thylakoid-bound SOD was 52000; the enzyme was composed of two equal subunits. Its activity was not sensitive to cyanide and hydrogen peroxide, and the isolated SOD contained Mn, but neither Fe nor Cu. Thus, the thylakoid-bound SOD is a Mn-containing enzyme. The subunit molecular weight of thylakoid Mn-SOD is the highest among Mn-SODs isolated so far, a fact which might reflect its binding to the membranes.     

In-vitro degradation of starch granules isolated from spinach chloroplasts

The initial reactions of transitory starch degradation in Spinacia oleracea L. were investigated using an in-vitro system composed of native chloroplast starch granules, purified chloroplast and non-chloroplast forms of phosphorylase (EC 2.4.1.1) from spinach leaves, and -amylase (EC 3.2.1.1) isolated from Bacillus subtilis. Starch degradation was followed by measuring the release of soluble glucans, by determining phosphorylase activity, and by an electron-microscopic evaluation following deep-etching of the starch granules. Starch granules were readily degraded by -amylase but were not a substrate for the chloroplast phosphorylase. Phosphorolysis and glucan synthesis by this enzyme form were strictly dependent upon a preceding amylolytic attack on the starch granules. In contrast, the non-chloroplast phosphorylase was capable of using starch-granule preparations as substrate. Hydrolytic degradation of the starch granules was initiated at the entire particle surface, independently of its size. As a result of amylolysis, soluble glucans were released with a low degree of polymerization. When assayed with these glucans as substrate, the chloroplast phosphorylase form exhibited a higher apparent affinity and a higher reaction velocity compared with the non-chloroplast phosphorylase form. It is proposed that transitory starch degradation in vivo is initiated by hydrolysis; phosphorolysis is most likely restricted to a pool of soluble glucan intermediates.Abbreviations Glc1P Glucose 1-phosphate - Mes 2(N-morpholino)ethanesulfonic acid - Pi Orthophosphate     

Light activation of fructose bisphosphatase in isolated spinach chloroplasts and deactivation by hydrogen peroxide

Spinach chloroplast fructose bisphosphatase (EC 3.1.3.11.) exists in both oxidised and reduced forms. Only the latter has the kinetic properties that allow it to function at physiological concentrations of fructose 1,6-bisphosphate and Mg²⁺. Illumination of freshly prepared type A chloroplasts causes a conversion of oxidised to reduced enzyme. The rate of this conversion does not limit the rate of CO₂ fixation. In the dark the reduced enzyme partially reverts back to the oxidised form. If catalase is omitted from the reaction medium the rate of CO₂ fixation by chloroplasts is decreased and seems to be limited by the rate of conversion of the enzyme to the reduced form. The physiological significance of the light dependent generation of dithiol compounds (such as thioredoxin) within chloroplasts is discussed.     

Calcium binding by spinach stromal proteins

Calcium binding to spinach (Spinacia oleracea L.) stromal proteins was examined by dual-wavelength spectrophotometry using the metallochromic indicator tetramethylmurexide. The data are consistent with the existence of at least two, probably independent, classes of binding sites. The total number of binding sites varied between 90–155 nmol·mg^–1 protein with average binding constants of 1.1–2.7·mM^–1. Both Mg²⁺ and La³⁺ inhibited calcium binding competitively, with average inhibitor constants of 0.26·mM^–1 and 39.4·mM^–1, respectively; an increase in the potassium concentration up to 50 mM had no effect. In a typical experiment a decrease in pH (7.8 to 7.1) resulted in a decrease in the total number of calcium binding sites from 90 to 59 nmol·mg^–1 protein, but in an increase of the average affinity from 2.7 to 4.5·mM^–1. Calculations, using these data and those of Gross and Hess (1974, Biochim. Biophys. Acta 339, 334–346) for binding site I of washed thylakoid membranes, showed that the free-Ca²⁺ concentration in the stroma under dark conditions, pH 7.1, is higher than under light conditions, pH 7.8. The physiological relevance of the observed calcium binding by stromal proteins is discussed.Abbreviations Ca _b ²⁺ bound calcium - Ca _f ²⁺ free calcium     

The effect of different temperatures on fatty-acid synthesis and polyunsaturation in cell suspension cultures

Cell suspension cultures of Catharanthus roseus G. Don, Glycine max (L.) Merr. and Nicotiana tabacum L. were incubated with [¹⁴C]acetate, [¹⁴C]oleic acid and [¹⁴C]linoleic acid at five different temperatures ranging from 15 to 35° C. When the incubation temperature was increased, [¹⁴C]acetate was incorporated preferentially into [¹⁴C]palmitate, with a concomitant drop in [¹⁴C]oleate formation. Between 15 and 20° C, [¹⁴C]oleic acid accumulated in C. roseus cells. In all cultures, optimum desaturation of [¹⁴C]oleic acid to [¹⁴C]linoleic acid occurred between 20 and 25° C, and in G. max this was also the optimal range for desaturation of [¹⁴C]linoleic acid to [¹⁴C]linolenic acid. Elongation of [¹⁴C]palmitic acid was inhibited when cultures grown at 15° C for 25 h were subsequently incubated with [¹⁴C]acetate at 25° C. [¹⁴C]oleic acid accumulated in G. max and C. roseus cultures grown at 35° C for 25 h and subsequently incubated at 25° C. Desaturation of [¹⁴C]oleic acid increased up to 25° C, but then decreased or leveled off depending on the cell line and on the temperature prior to incubation.     

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate) or [¹⁴C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.Abbreviastions Chlorophyll a_GC Chlorophyll a, esterified with geranylgeraniol - GGPP geranylgeranyl pyrophosphate - HPLC high pressure liquid chromatography - IPP isopentenyl pyrophosphate     

Metabolite levels in the stroma of spinach chloroplasts exposed to osmotic stress: Effects of the pH of the medium and exogenous dihydroxyacetone phosphate

The levels of stromal photosynthetic intermediates were measured in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Stressed chloroplasts showed slower rates of metabolite accumulation upon illumination than controls. Relative to other metabolites sedoheptulose-1,7-bisphosphate (SBP) and fructose-1,6-bisphosphate (FBP) accumulated in the stroma in the stressed treatments. Under these conditions 3-phosphoglycerate (3-PGA) efflux to the medium was restricted. Chloroplasts previously incubated with [³²P]KH₂PO₄ and [³²P]dihydroxyacetone phosphate ([³²P]DAP) in the dark were characterized by very high FBP and SBP levels prior to illumination. Metabolism of these pools upon illumination increased with increasing pH of the medium but was consistently inhibited in osmotically stressed chloroplasts. The responses of stromal FBP and SBP pools under hypertonic conditions are discussed in terms of both inhibited light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37), and likely increases in stromal ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) active-site concentrations.Abbreviations and symbols DAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - _s osmotic potential     

Characterization of calcium fluxes across the envelope of intact spinach chloroplasts

Calcium fluxes across the envelope of intact spinach chloroplasts (Spinacia oleracea L.) in the light and in the dark were investigated using the metallochromic indicator arsenazo III. Light induces Ca²⁺ influx into chloroplasts. The action spectrum of light-induced Ca²⁺ influx and the inhibitory effect of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) indicate an involement of photosynthetic electron transport in this process. The driving force for light-induced Ca²⁺ influx is most likely a change in the membrane potential component of the proton motive force. This was demonstrated by the use of agents modifying the membrane potential (lipophilic cations, ionophores, different KCl concentrations). The activation energy of the observed Ca²⁺ influx is about 92 kJ mol^-1. Verapamil and nifedipine, two Ca²⁺-channel blockers, have no inhibitory effect on light-induced Ca²⁺ influx, but enhance ferricyanide-dependent oxygen evolution. Inhibition of Ca²⁺ influx by ruthenium red reduces the light-dependent decrease in stromal NAD⁺ level.Abbreviations and symbols Chl chlorophyll - DCMU 3-(3',4'-dichlorophenyl)-1,1-dimethylurea - FCCP earbonyl cyanide p-trifluoromethoxyphenylhydrazone - PGA 3-phosphoglyceric acid - ABA⁺ tetrabutylammonium chloride - TPP⁺ tetraphenylphosphonium chloride - E membrane potential     

The formation of homogentisate in the biosynthesis of tocopherol and plastoquinone in spinach chloroplasts

Homogentisate is the precursor in the biosynthesis of -tocopherol and plastoquinone-9 in chloroplasts. It is formed of 4-hydroxyphenylpyruvate of the shikimate pathway by the 4-hydroxyphenylpyruvate dioxygenase. In experiments with spinach the dioxygenase was shown to be localized predominatedly in the chloroplasts. Envelope membranes exhibit the highest specific activity, however, because of the high stromal portion of chloroplasts, 60–80% of the total activity is housed in the stroma. The incorporation of 4-hydroxyphenylpyruvate into 2-methyl-6-phytylquinol as the first intermediate in the tocopherol synthesis by the two-step-reaction: 4-Hydroxyphenylpyruvate Homogentisate 2-Methyl-6-phytylquinol was demonstrated by using envelope membranes. Homogentisate originates directly from 4-hydroxyphenylpyruvate of the shikimate pathway. Additionally, a bypass exists in chloroplasts which forms 4-hydroxyphenylpyruvate from tyrosine by an L-amino-acid oxidase of the thylakoids and in peroxisomes by a transaminase reaction. Former results about the dioxygenase in peroxisomes were verified.     

Intracellular localization of serine acetyltransferase in spinach leaves

Intact chloroplasts isolated from spinach leaves by a combination of differential and Percoll density gradient centrifugation and free of mitochondrial and peroxisomal contamination contained about 35% of the total leaf serine acetyltransferase (EC 2.3.1.30) activity. No appreciable activity of the enzyme could be detected in the gradient fractions containing broken chloroplasts, mitochondria, and peroxisomes. L-cysteine added to the incubation mixture at 1 mM almost completely inhibited serine acetyltransferase activity, both of leaf and chloroplast extracts. D-cysteine was much less inhibitory. L-cystine up to 5 mM and O-acetyl-L-serine up to 10 mM had no effect on the enzyme activity. When measured at pH 8.4, the enzyme extracted from the leaves had a K _m for L-serine of 2.4, the enzyme from the chloroplasts a K _m of 2.8 mM.Abbreviations NAS N-acetyl-L-serine - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - 3-PGA D-3-phosphoglycerate - SATase serine acetyltransferase     

Fatty-acid synthesis in chloroplasts from mustard (Sinapis alba L.) cotyledons: formation of acetyl coenzyme A by intraplastid glycolytic enzymes and a pyruvate dehydrogenase complex

Chloroplasts from the cotyledons of mustard (Sinapis alba L.) seedlings were isolated on Percoll gradients, and showed a high degree of intactness (92%) and purity as judged by electron microscopy and marker-enzyme analysis (cytoplasmic contamination lower than 0.4% on a protein basis). The chloroplasts synthesized longchain fatty acids from both precursors [1-¹⁴C] acetate and [2-¹⁴C]pyruvate; maximum incorporation rates were 96 nmol·(mg Chl)^-1·h^-1 for acetate and 213 nmol·(mg Chl)^-1·h^-1 for pyruvate. Acetyl-CoA-producing enzymatic activities, namely acetyl-CoA synthetase (EC 6.2.1.1.) and a pyruvate dehydrogenase complex, showed specific activities of 14.8 nmol·(mg protein)^-1·min^-1 and 18.2 nmol·(mg protein)^-1·min^-1, respectively. The glycolytic enzymes phosphoglyceromutase (EC 2.7.5.3) phosphopyruvate hydratase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were all found to be components of these chloroplasts, thus indicating a possible pathway for intraplastid acetyl-CoA formation.Abbreviations ACS acetyl coenzyme A synthetase - Chl chlorophyll - DTE 1,4-dithioerythritol - PDHC pyruvate dehydrogenase complex - 3-PGA 3-phosphoglyceric acid     

Subcellular volumes and metabolite concentrations in spinach leaves

Cellular and subcellular volumes in mature leaves of spinach (Spinacia oleracea L. US Hybrid 424) were determined stereologically from light and electron micrographs. Forty-nine-day-old leaves of spinach with a total leaf volume of 1177 μL per mg chlorophyll (Chl) were found to be composed of 3% epidermis, 58% mesophyll, 1% vascular tissue, 5% apoplasm and 32% gas space. In the epidermal cells 89% of the volume was occupied by the vacuole. The mesophyll cells consisted, expressed in mg·Chl⁻¹, of 546 μL (79%) vacuole, 66 μL (9.5%) chloroplast stroma, 24 μL (34%) cytosol, 3.7 μL (0.5%) mitochondria and 2.1 μL (0.3%) nucleus. From previous measurements of the subcellular levels of sucrose, of phosphorylated intermediates of carbohydrate metabolism, of malate, oxoglutarate and various amino acids in illuminated leaves, and the above subcellular volumes, the corresponding subcellular metabolite concentrations have been determined. Of the substances measured, only with malate was the concentration higher in the vacuole than in the cytosol. The concentration of sucrose in the cytosol was 5 times, and that of amino acids even 30 times higher than in the vacuole.     

Membrane lipids in heat injury of spinach chloroplasts

Heat treatment of intact leaves and of isolated thylakoid membranes from spinach (Spinacia oleracea L. cvs. Monatol and Montako) caused inactivation of photochemical processes such as electron transport through photosystem II and photophos-phorylation. Membrane lipid analysis demonstrated that heat-induced damage to thylakoids is not caused by chemical alterations in the lipids such as oxidation of unsaturated fatty acids, or release of free fatty acids due to hydrolysis of lipids. Partial extraction of lipids from isolated chloroplast membranes before and after thermal inactivation do not point to drastic changes in the binding relations of the lipids within the membranes. However, it cannot be excluded that during high temperature treatment changes in lipid-lipid interactions and/or delocalization of specific lipids within the thylakoids might be responsible for the disorganization of the functional integrity of the membranes. Since thermostability of chloroplast membranes is decreased when they are exposed to free unsaturated fatty acids, small amounts of membrane lipids which become hydrolyzed during extended heat treatment may partly contribute to primary heat damage.