The role of the triose-phosphate shuttle and glycolytic intermediates in fatty-acid and glycerolipid biosynthesis in pea root plastids

The capacity of the triose-phosphate shuttle and various combinations of glycolytic intermediates to substitute for the ATP requirement for fatty-acid and glycerolipid biosynthesis in pea (Pisum sativum L.) root plastids was assessed. In all cases, ATP gave the greatest rates of fatty-acid and glycerolipid biosynthesis. Rates of up to 66 and 27 nmol·(mg protein)^–1·h^–1 were observed for the incorporation of acetate and glycerol-3-phosphate into lipids in the presence of ATP. In the absence of exogenously supplied ATP, the triose-phosphate shuttle gave up to 44 and 33% of the ATP-control activity in promoting fatty-acid and glycerolipid biosynthesis from acetate and glycerol-3-phosphate, respectively. The optimum shuttle components were 2 mM dihydroxyacetonephosphate (DHAP), 2 mM oxaloacetic acid and 4 mM inorganic phosphate (referred to as the DHAP shuttle). Glyceraldehyde-3-phosphate, as a shuttle triose, was approximately 82% as effective as DHAP in promoting fatty-acid synthesis while 2-phosphoglycerate, 3-phosphoglycerate, and phosphoenolpyruvate were only 27–37% as effective as DHAP. When glycolytic intermediates were used as energy sources for fatty-acid synthesis, in the absence of both exogenously supplied ATP and the triose-phosphate shuttle, phosphoenolpyruvate, 2-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate each gave 48%, 17%, 23% and 17%, respectively, of the ATP-control activity. Other triose phosphates tested were much less effective in promoting fatty-acid synthesis. When exogenously supplied ATP was supplemented with the DHAP shuttle or glycolytic intermediates, the complete shuttle increased fatty-acid biosynthesis by 37% while DHAP alone resulted in 24% stimulation. Glucose-6-phosphate, fructose-6-phosphate and glycerol-3-phosphate similarly all improved the rates of fatty-acid synthesis by 20–30%. In contrast, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate all inhibited fatty-acid synthesis by approximately 10% each. The addition of the DHAP shuttle and glycolytic intermediates with or without exogenously supplied ATP caused an increase in the proportion of radioactive oleate and a decrease in the proportion of radioactive palmitate synthesized. The use of these alternative energy sources resulted in higher amounts of free fatty acids and triacylglycerol, and lower amounts of diacylglycerol and phosphatidic acid. The data presented here indicate that ATP is superior in promoting in-vitro fatty-acid biosynthesis in pea root plastids; however, both the triose-phosphate shuttle and glycolytic metabolism can produce some of the ATP required for fatty-acid biosynthesis in these plastids.Abbreviations DHAP dihydroxyacetonephosphate - Fru6P fructose-6-phosphate - G3P glycerol-3-phosphate - Glc6P glucose-6-phosphate - OAA oxaloacetate - PEP phosphoenolpyruvate - 2PGA 2-phosphoglycerate - 3PGA 3-phosphoglycerate - 3PGalde glyceraldehyde-3-phosphate This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada.     

Fatty acid synthesis by isolated leucoplasts from developingBrassica seeds: Role of nucleoside triphosphates and DHAP-shuttle as the source of energy

Fatty acid synthesis in leucoplasts isolated from developing seeds ofBrassica campestris was absolutely dependent on external source of ATP. None of the other nucleoside triphosphates could replace ATP in the reaction mixture. Use of ADP alone also resulted in reduced rates of fatty acid synthesis. However, in combination with inorganic phosphate or inorganic pyrophosphate, it improved the rate of fatty acid synthesis, giving up to 50% of the ATP-control activity. Inorganic phosphate or inorganic pyrophosphate alone again did not serve as an energy source for fatty acid synthesis. AMP, alongwith inorganic pyrophosphate could promote fatty acid synthesis to up to 42% of the activity obtained with ATP. The three components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate of dihydroxy acetone phosphate-shuttle together could restore 50% of the activity obtained with ATP. Omission of any one of the components of this shuttle drastically reduced the rate of fatty acid synthesis to 15–24% of the ATP-control activity. Inclusion of ATP in reaction mixtures containing shuttle components enhanced the rate of synthesis over control. The optimum ratio of shuttle components dihydroxy acetone phosphate, oxaloacetic acid, inorganic phosphate determined was 1:1:2. Maximum rates of fatty acid synthesis were obtained when dihydroxy acetate phosphate was used as the shuttle triose. Glyceraldehyde-3-P, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate as shuttle trioses were around 35–60% as effective as dihydroxy acetone phosphate in promoting fatty acid synthesis. The results presented here indicate that although the isolated leucoplasts readily utilize exogenously supplied ATP for fatty acid synthesis, intraplastidic ATP could also arise from dihydroxy acetone phosphate shuttle components or other appropriate metabolites     

The Utilization of Glycolytic Intermediates as Precursors for Fatty Acid Biosynthesis by Pea Root Plastids

Radiolabeled pyruvate, glucose, glucose-6-phosphate, acetate, and malate are all variously utilized for fatty acid and glycerolipid biosynthesis by isolated pea (Pisum sativum L.) root plastids. At the highest concentrations tested (3-5mM), the rates of incorporation of these precursors into fatty acids were 183, 154, 125, 99 and 57 nmol h-1 mg-1 protein, respectively. In all cases, cold pyruvate consistently caused the greatest reduction, whereas cold acetate consistently caused the least reduction, in the amounts of each of the other radioactive precursors utilized for fatty acid biosynthesis. Acetate incorporation into fatty acids was approximately 55% dependent on exogenously supplied reduced nucleotides (NADH and NADPH), whereas the utilization of the remaining precursors was only approximately 10 and 20% dependent on added NAD(P)H. In contrast, the utilization of all precursors was greatly dependent (85-95%) on exogenously supplied ATP. Palmitate, stearate, and oleate were the only fatty acids synthesized from radioactive precursors. Higher concentrations of each precursor caused increased proportions of oleate and decreased proportions of palmitate synthesized. Radioactive fatty acids from all precursors were incorporated into glycerolipids. The data presented indicate that the entire pathway from glucose, including glycolysis, to fatty acids and glycerolipids is operating in pea root plastids. This pathway can supply both carbon and reduced nucleotides required for fatty acid biosynthesis but only a small portion of the ATP required     

Characterization of the Glycerolipid Composition and Biosynthetic Capacity of Pea Root Plastids

The glycerolipid composition of pea (Pisum sativum L.) root plastids and their capacity to synthesize glycerolipids from [UL-14C]glycerol-3-phosphate were determined. Pea root plastids primarily consist of monogalactosyldiacylglycerol, triacylglycerol, phosphatidylcholine, digalactosyldiacylglycerol, and diacylglycerol. Maximum rates of total glycerolipid biosynthesis were obtained in the presence of 2.4 mM glycerol-3-phosphate, 15 mM KHCO3, 0.2 mM sodium-acetate, 0.5 mM each of NADH and NADPH, 0.05 mM coenzyme A, 2 mM MgCl2, 1 mM ATP, 0.1 M Bis-Tris propane (pH 7.5), and 0.31 M sorbitol. Glycerolipid biosynthesis was completely dependent on exogenously supplied ATP, coenzyme A, and a divalent cation, whereas the remaining cofactors improved their activity from 1.3- to 2.4-fold. Radioactivity from glycerol-3-phosphate was recovered predominantly in phosphatidic acid, phosphatidylglycerol, diacylglycerol, and triacylglycerol with lesser amounts in phosphatidylcholine and monoacylglycerol. The proportions of the various radiolabeled lipids that accumulated were dependent on the pH and the concentration of ATP and glycerol-3-phosphate. The data presented indicate that pea root plastids can synthesize almost all of their component glycerolipids and that glycerolipid biosynthesis is tightly coupled to de novo fatty acid biosynthesis. pH and the availability of ATP may have important roles in the regulation of lipid biosynthesis at the levels of phosphatidic acid phosphatase and in the reactions that are involved in phosphatidylglycerol and triacylglycerol biosynthesis.     

Photosynthesis by isolated pea chloroplasts: some effects of adenylates and inorganic pyrophosphate

When added singly to chloroplasts isolated from young pea (Pisum sativum) leaves, both inorganic pyrophosphate (PPi) and small quantities (0.2 mm) of ADP inhibit photosynthesis, but when added together they cause a marked stimulation. ATP (at 0.2 mm) is less inhibitory (or not inhibitory) when added alone, but like ADP, stimulates when added in the presence of PPi. This behavior is in marked contrast to that of spinach (Spinacia oleracea) chloroplasts which are normally stimulated rather than inhibited by PPi and which are largely unresponsive to exogenous adenylates. The inhibitory behavior of PPi with pea chloroplasts was observed under conditions where external hydrolysis to Pi is negligible. It is proposed that the exchange of organic and PPi across the chloroplast envelope may be more rapid in chloroplasts from young pea leaves than in chloroplasts from spinach and that interaction between these two processes could account for the principal observations.     

A preliminary analysis of Fatty Acid synthesis in pea roots

Subcellular fractions from pea (Pisum sativum L.) roots have been prepared by differential centrifugation techniques. Greater than 50% of the recovered plastids can be isolated by centrifugation at 500g for 5 minutes. Plastids of this fraction are largely free from mitochondrial and microsomal contamination as judged by marker enzyme analysis. De novo fatty acid biosynthesis in pea roots occurs in the plastids. Isolated pea root plastids are capable of fatty acid synthesis from acetate at rates up to 4.3 nanomoles per hour per milligram protein. ATP, bicarbonate, and either Mg²⁺ or Mn²⁺ are all absolutely required for activity. Coenzyme A at 0.5 millimolar improved activity by 60%. Reduced nucleotides were not essential but activity was greatest in the presence of 0.5 millimolar of both NADH and NADPH. The addition of 0.5 millimolar glycerol-3-phosphate increased activity by 25%. The in vitro and in vivo products of fatty acid synthesis from acetate were primarily palmitate, stearate, and oleate, the proportions of which were dependent on experimental treatments. Fatty acids synthesized by pea root plastids were recovered in primarily phosphatidic acid and diacylglycerol or as water soluble derivatives and the free acids. Lesser amounts were found in phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and monogalactosyldiacylglycerol.     

Lipid biosynthesis by isolated plastids from greening pea, Pisum sativum

Isolated etioplasts from 8-day-old dark-grown pea seedlings incorporated [1-(14)C]acetate into lipid at a relatively low rate. Plastids from seedlings that had been illuminated for at least 2 hr showed an enhanced incorporation provided the plastids were illuminated during incubation with the labeled acetate. Dark incubation or the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased the acetate-incorporating activity of the developing chloroplasts to the level observed with etioplasts. Light had a marked effect on the type of fatty acid into which acetate was incorporated by the developing chloroplasts. Unsaturated fatty acids (mostly oleic acid) accounted for 60-80% of the incorporated label if the plastids were illuminated, but in the dark or in the presence of DCMU the unsaturated acids accounted for only 0-15% of the label incorporated into lipid. The effect of ATP on incorporation was dependent on the maturity of the chloroplasts; mature pea chloroplasts were inhibited by ATP, whereas in developing plastids there was a slight stimulation by ATP. Inhibition of acetate incorporation into lipid by DCMU appears to be due to inhibition of noncyclic phosphorylation. Incorporation was restored by reduced 2,3,5,6-tetramethylphenylenediamine, which restored phosphorylation, but not by reduced N,N,N',N'-tetramethylphenylenediamine.     

Pyruvate dehydrogenase complex and acetyl-CoA carboxylase in pea root plastids: their characterization and role in modulating glycolytic carbon flow to fatty acid biosynthesis

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase(ACC, EC 6.4.1.2 [EC] ) have been characterized in pea root plastids.PDC activity was optimum in the presence of 1.0 mM pyruvate,1.5 mM NAD⁺ 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCl₂, 3.0 mM cysteine-HCl,and 0.1 M Tricine (pH 8.0) and represents approximately 47%of the total cellular activity. ACC activity was greatest inthe presence of 1.0 mM acetyl-CoA, 4 mM NaHCO₃ mM ATP, 10 mMMgCl₂, 2.5 mM dithiothreitol, and 100 mM Tricine (pH 8.0). Bothenzymes were stimulated by reduced sulphydryl reagents and inhibitedby sulphydryl inhibitors. ACC was also inhibited by malonyl-CoAwhile PDC was inhibited by both malonyl-CoA and NADH. Both enzymeswere stimulated by DHAP and UDP-galactose while ACC was alsostimulated by PEP and F₁,6P. Palmitic acid and oleic acid bothinhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoAinhibited both enzymes while PA and Lyso-PA inhibited PDC, butstimulated ACC. The results presented support the hypothesisthat PDC and ACC function in a co-ordinated fashion to promoteglycolytic carbon flow to fatty acid biosynthesis in pea rootplastids. Key words: Pisum sativum L., pyruvate dehydrogenase complex, acetyl-CoA carboxylase, roots, non-photosynthetic plastids     

Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos

The aim of this work was to investigate the partitioning of imported glucose 6-phosphate (Glc6P) to starch and fatty acids, and to CO₂ via the oxidative pentose phosphate pathway (OPPP) in plastids isolated from developing embryos of oilseed rape (Brassica napus L.). The ability of the isolated plastids to utilize concurrently supplied substrates and the effects of these substrate combinations on the Glc6P partitioning were also assessed. The relative fluxes of carbon from Glc6P to starch, fatty acids, and to CO₂ via the OPPP were close to 2∶1∶1 when Glc6P was supplied alone. Under these conditions NADPH generated via the OPPP was greater than that required by the concurrent rate of fatty acid synthesis. Fatty acid synthesis was unaffected by the presence or absence of exogenous NADH and/or NADPH and the requirement of fatty acid synthesis for reducing power is therefore met entirely by intraplastidial metabolism. When Glc6P was supplied in the presence of either pyruvate or pyruvate and acetate, the total flux from these metabolites to fatty acids was up to threefold greater than that from either Glc6P or pyruvate when they were supplied singly. In these experiments there was little competition between Glc6P and pyruvate in fatty acid synthesis and the flux to starch was unchanged. This implies that the starch and fatty acid biosynthesis pathways did not compete for the exogenously supplied ATP on which they were strongly dependent. When Glc6P and pyruvate were provided together, the NADPH generated by the OPPP pathway was less than that required by the concurrent rate of fatty acid synthesis. This suggests that the metabolism of exogenous Glc6P via the OPPP can contribute to the NADPH demand created during fatty acid synthesis but it also indicates that other intraplastidial sources of reducing power must be available under the in-vitro conditions used.     

Pyrophosphate inhibition of carbon dioxide fixation in isolated pea chloroplasts by uptake in exchange for endogenous adenine nucleotides

Carbon dioxide-dependent O(2) evolution by isolated pea (Pisum sativum) chloroplasts was inhibited by inorganic pyrophosphate (PPi). Oxygen evolution was also inhibited by high concentrations of orthophosphate (Pi) and the inhibition was relieved by 3-phosphoglycerate. In contrast, the inhibition by PPi was not relieved by 3-phosphoglycerate, indicating that hydrolysis of PPi and accumulation of inhibitory concentrations of Pi were not occurring. In agreement with this suggestion, the percentage of (14)C-labeled products diffusing out of the chloroplasts was increased by Pi but not by PPi. The inhibition of O(2) evolution by PPi was reversed by ATP. The concentration of PPi required for 50% inhibition was 1.2 to 1.4 mm and the subsequent stimulation by ATP was half-maximal at 16 to 25 mum. Carbon dioxide-dependent O(2) evolution by spinach chloroplasts, or chloroplasts isolated from older pea plants, was not significantly inhibited by PPi.Chloroplasts were preloaded with (14)C-ATP and release of the labeled nucleotides was measured to assess the activity of adenine nucleotide transport across the inner chloroplast envelope membrane. A rapid exchange was promoted by the addition of exogenous ATP. Addition of PPi also resulted in a release of endogenous nucleotides. We suggest that PPi inhibits CO(2) fixation by entering the chloroplast in exchange for endogenous adenine nucleotides via the transporter on the inner envelope membrane. The subsequent depletion of the internal adenine nucleotide pool would result in decreased CO(2) fixation due to insufficient ATP. Addition of ATP to PPi-inhibited chloroplasts apparently results in uptake of catalytic amounts of ATP and restoration of the internal adenine nucleotide pool thus relieving the inhibition of CO(2) fixation.     

Secretion, subcellular localization and metabolic status of inorganic pyrophosphate in human platelets. A major constituent of the amine-storing granules.

The platelet content of PPi is 1.90 +/- mumol/10(11) platelets (S.E.M., n = 19) or about 10.5 nmol/mg of protein, several hundred times that found for rat liver. Some 80% of this PPi is secreted by platelets treated with thrombin with a time course and dose-response relationship similar to secretion of ATP, ADP and 5-hydroxytryptamine (serotonin) from the platelet dense granules. During platelet aggregation induced by ADP and adrenaline, substantial amounts of PPi were secreted, but no release of acid hydrolases was observed. Subcellular-fractionation studies showed that the PPi is highly enriched in the same fraction that contains the storage organelles which store ATP, ADP, Ca2+ and 5-hydroxytryptamine. Inorganic pyrophosphatase was present mainly in the soluble fraction and in the mitochondria. Secretion studies done with platelets prelabelled with [32P]Pi showed that the sequestered PPi was relatively metabolically inactive, as is the ATP and ADP in the storage organelles. The possible participation of PPi in the formation of a bivalent-cation-nucleotide complex associated with amine storage is discussed.     

Photosynthesis by isolated protoplasts, protoplast extracts, and chloroplasts of wheat: influence of orthophosphate, pyrophosphate, and adenylates

Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat (Triticum aestivum) have rates of photosynthesis as measured by light-dependent O₂ evolution of about 100 to 150 micromoles of O₂ per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO₃ with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O₂ per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset.

Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP.

Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO₃ and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

     

Submitochondrial localization and function of alkaline inorganic pyrophosphatase in maize seedlings.

Alkaline inorganic pyrophosphatase and Mg-ATPase are localized within the mitoplast of maize seeding mitochondria. NaF inhibited the PPase activity, whereas oligomycin and dicyclohexylcarbodiimide inhibited the Mg-ATPase activity. The mitoplast preparation synthesized PPi from Pi under conditions excluding hydrolysis of endogenous ATP. PPi synthesis was inhibited by ADP, antimycin A, NaCN and 2,4- dinitrophenol but not by oligomycin. It is suggested that PPi synthesis in the maize seedling mitochondria proceeds at the expense of the energy of electron transport chain and is independent of the ATP synthesis.     

Transport of inorganic phosphate and C3- and C6-sugar phosphates across the envelope membranes of potato tuber amyloplasts

Amyloplasts have been isolated from tubers of potato plants (Solarium tuberosum. cv. Desirée). As it is difficult to isolate amyloplasts that have a high starch content, we used transformed plants in which the content of starch was reduced. This was achieved by decreasing the activity of ADP-glucose pyrophosphorylase by antisense techniques (Müller-Röber et al., 1992, EMBO. 11, 1229–1238). In the isolated plastids the activity of glutamine-oxoglutarate-aminotransferase (glutamate synthase, EC 2.6.1.53) was dependent upon the intactness of the plastids. For the supply of redox equivalents the addition of glucose-6-phosphate (Glc6P) was required. Glucose-1-phosphate (Glc1P) did not support glutamate synthesis. Plastids were treated with Triton X-100 and the solubilized proteins reconstituted into liposomes. Transport measurements with these liposomes revealed that inorganic phosphate (Pi), dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate and Glc6P are transported in a counter-exchange mode. Transport of phosphoenolpyruvate was low and Glc1P was virtually not transported in exchange for Pi. Kinetic constants were determined for the Pi/Pi and Glc6P/Pi counter exchanges. For comparison, proteins of mitochondria from potato tubers and pea leaves were reconstituted into liposomes. As expected, the Pi/Pi exchange across the mitochondrial membrane was not affected by DHAP and Glc6P. Kinetic constants of the Pi/Pi counter exchange were determined for potato tuber mitochondria.Abbreviations DHAP dihydroxyacetone phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP Phosphoenolpyruvate - 3-PGA 3-phosphoglycerate - Pi inorganic phosphate - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl] glycine This work was supported by Deutsche Forschungsgemeinschaft.     

Phosphate free perfusion prevents washout of tissue creatine in Langendorff perfused rabbit heart.

Rabbit hearts were perfused with Krebs-Henseleit bicarbonate buffer supplemented with 15 mM glucose and 10 mU/ml of insulin +/- Pi. At the end of 60 min the hearts were freeze-clamped and the content of ATP, creatine phosphate, creatine, lactate, pyruvate, DHAP and 3-P glycerate were determined enzymatically in neutralized perchloric acid tissue extracts. The free cytosolic ADP and Pi and the cytosolic NAD+ redox and phosphorylation potentials were calculated from the measured metabolite concentrations. Pi free perfusion resulted in increased creatine, free cytosolic ADP and cytosolic phosphorylation potential, decreased calculated free Pi and no change in cardiac ATP and creatine phosphate content. The increase in the cytosolic phosphorylation potential was due to the lowering of cytosolic free Pi. The increase in ADP was due to the increase in creatine. The increase in creatine appeared to be due to an inhibition of creatine efflux from the heart during Pi free perfusion which was mediated by an enhanced Na+ electrochemical gradient.     

Pyrophosphate import and synthesis by plant mitochondria

The matrix level of pyrophosphate (PPi) in mitochondria isolated from etiolated pea ( Pisum sativum L. cv. Alaska) stems was evaluated, on the basis of an enzymatic assay, to be approx. 0.2 m M . Pyrophosphate could enter from the cytoplasm to the mitochondria via adenine nucleotide translocase (ANT), because F^– and Ca²⁺ (two penetrating PPiase inhibitors) and atractylate (ANT inhibitor) inhibited PPiase activity in isolated mitochondria supplied with PPi. This result was also confirmed by measuring oxygen consumption and membrane potential (ΔΨ) in succinate-energized mitochondria. In a medium free of phosphate (Pi), the addition of PPi before the substrate rendered possible an ADP-stimulated oxygen consumption that was inhibited by F^– or Ca²⁺. In a similar experiment, ADP induced the dissipation of ΔΨ when it was added after the succinate-generated ΔΨ had reached a steady state and, again, F^– inhibited this dissipation. These results imply that PPi enters the mitochondria where it is hydrolyzed to 2 Pi which become available for the H⁺-ATPase (EC 3.6.1.34). In addition, PPi may be synthesized by the H⁺-PPiase (EC 3.6.1.1), acting as a synthase. This evidence arises from the observation that Pi stimulated an oxygen consumption (respiratory control ratio of 1.7) that was inhibited by F^– or Ca²⁺. The physiological role of the mitochondrial H⁺-PPiase is discussed in the light of the consideration that this enzyme can catalyse a readily reversible reaction.     

Characterization of Fatty Acid biosynthesis in isolated pea root plastids

Fatty acid biosynthesis from Na[1-¹⁴C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl₂, 1 millimolar each of MnCl₂ and glycerol-3-phosphate, 15 millimolar KHCO₃, 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35°C. At the standard incubation temperature of 25°C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [¹⁴C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.     

ADP/ATP Translocator from Pea Root Plastids (Comparison with Translocators from Spinach Chloroplasts and Pea Leaf Mitochondria)

The kinetic properties of the adenosine 5[prime]-diphosphate/adenosine 5[prime]-triphosphate (ADP/ATP) translocator from pea (Pisum sativum L.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea L.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. The Km(ATP) value of the pea root ADP/ATP translocator was 10 [mu]M and that for ADP was 46 [mu]M. Corresponding values of the spinach ADP/ATP translocator were 25 [mu]M and 28 [mu]M, respectively. Comparable results were obtained for the reconstituted ATP transport activities. The transport was highly specific for ATP and ADP. Adenosine 5[prime]-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake. With pea root plastids and spinach chloroplasts, Km values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.     

Compartmentation of ATP:citrate lyase in plants

Extracts prepared from young leaves of Pea (Pisum sativum), tobacco (Nicotiana tabacum), rape (Brassica napus), and spinach (Spinacia oleracea) all contained ATP:citrate lyase (ACL) activity, which was most active in rape leaflets (130 nmol min(-1) g fresh weight). In rape and spinach, ACL activity was predominantly localized in the plastids (between about 78% and 90% of the total activity), whereas in pea and tobacco, distribution was mainly cytosolic (about 85% and 78%, respectively, of the total). These distributions were calculated from the relative distributions of plastid and cytosol marker enzymes. Cross-reactivity between plant and rat ACL antibody was carried out by immunoblot analysis and, in rape and spinach, showed that a 120-kD protein, presumably indicating homomeric ACL proteins, was present in both cytosolic and plastidic fractions. In pea, two cross-reacting proteins were detected, the major material being in the cytosol fraction. Therefore, ACL occurs both in the cytosol and plastids of higher plants, but the distribution of activity changes according to the species. The plastidic ACL is proposed to function for the supply of acetyl-coenzyme A for lipid biosynthesis de novo, whereas the cytosolic ACL may provide acetyl-coenzyme A for the mevalonate pathway or fatty acid elongation.     

Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.)

The aim of this work was to investigate the capacity for synthesis of starch and fatty acids from exogenous metabolites by plastids from developing embryos of oilseed rape (Brassica napus L.). A method was developed for the rapid isolation from developing embryos of intact plastids with low contamination by cytosolic enzymes. The plastids contain a complete glycolytic pathway, NADP-glucose-6-phosphate dehydrogenase, NADP-6-phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase, NADP-malic enzyme, the pyruvate dehydrogenase complex (PDC), and acetyl-CoA carboxylase. Organelle fractionation studies showed that 67% of the total cellular PDC activity was in the plastids. The isolated plastids were fed with ¹⁴C-labelled carbon precursors and the incorporation of ¹⁴C into starch and fatty acids was determined. ¹⁴C from glucose-6-phosphate (G-6-P), fructose, glucose, fructose-6-phosphate and dihydroxyacetone phosphate (DHAP) was incorporated into starch in an intactness- and ATP-dependent manner. The rate of starch synthesis was highest from G-6-P, although fructose gave rates which were 70% of those from G-6-P. Glucose-1-phosphate was not utilized by intact plastids for starch synthesis. The plastids utilized pyruvate, G-6-P, DHAP, malate and acetate as substrates for fatty acid synthesis. Of these substrates, pyruvate and G-6-P supported the highest rates of synthesis. These studies show that several cytosolic metabolites may contribute to starch and/or fatty acid synthesis in the developing embryos of oilseed rape.