Activation and Deactivation of H-ATPase in Intact Chloroplasts

The light activation mechanism of the latent H⁺-ATPase was investigated in intact spinach (Spinacia oleracea, Hybrid 424) chloroplasts. The following observations were made. (a) Photosystem I electron acceptors such as methyl viologen, nitrite, oxaloacetate, etc., inhibit the light activation of the enzyme. (b) The electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) fully inhibits the process. (c) Ascorbate plus diaminodurene or dithionite can restore light activation in DCMU-poisoned chloroplasts. (d) The activated state of the enzyme decays rather slowly (within a few minutes) after illumination of the intact chloroplasts. (e) The rate of dark decay is accelerated by oxidants (H₂O₂ or ferricyanide) and slowed down by dithiothreitol.

It is suggested that the physiological mechanism for regulation of the H⁺-ATPase involves oxidation and reduction reactions in a manner which resembles the regulation of the light-activated carbon cycle enzymes.

     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Photosynthetic characteristics of chloroplasts isolated fromMesembryanthemum crystallinum L., a halophilic plant capable of Crassulacean acid metabolism

Photosynthetically highly active chloroplasts were routinely obtained by rupture of leaf protoplasts from the halophyteMesembryanthemum crystallinum which exhibited the photosynthetic characteristics of either a C₃ plant when grown with 20 mmol l^-1 NaCl in the rooting medium, or a Crassulacean-acid-metabolism (CAM) plant when grown with 400 mmol l^-1 NaCl. Photosynthesis rates of C₃ and CAM chloroplasts were 150–250 and 90–150 μmol mg^-1 chlorophyll h^-1, respectively. Because of osmotic adjustment, CAM chloroplasts required higher sorbitol concentrations (0.7–0.8 mol l^-1) in the assay medium than C₃ chloroplasts (0.3–0.4 mol l^-1) for optimum activity. Substitution of sorbitol by NaCl as the osmoticum strongly reduced photosynthesis of CAM chloroplasts. Rates of electron transport (ferricyanide reduction, uncoupled) remained unaffected over a range of sorbitol concentrations (0 to 1 mol l^-1). Sensitivity of electron transport to increasing levels of NaCl was less pronounced than the NaCl-sensitivity of CO₂ fixation by intact chloroplasts. The CAM chloroplasts showed a broad pH optimum of photosynthesis between pH 7.0 and 8.2; photosynthesis of C₃ chloroplasts dropped markedly below pH 7.6. The CAM chloroplasts maintained a higher transenvelope proton gradient than C₃ chloroplasts both in the light and dark. External pyruvate (5 mmol l^-1) inhibited photosynthesis of CAM chloroplasts, but not of C₃ chloroplasts. Inhibition was reduced by increased external concentrations of orthophosphate.     

The inhibition of photosynthetic electron transport by methyl parathion

The effect of methyl parathion (metacid-50), an organophosphorous insecticide, on the Hill reactions of isolated mesophyll chloroplasts ofSorghum vulgare was studied. The pesticide was found to inhibit the Hill reaction with all the Hill oxidants tested, namely potassium ferricyanide,2,6-dichlorophenol indophenol and para-benzoquinone. The concentration of the pesticide required to inhibit 50% of the control Hill activity (I₅₀value) was found to vary with the different Hill oxidants.     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

Different selectivities of oxidants during oxidation of methionine residues in the α-1-proteinase inhibitor

Oxidation of the reactive site methionine (Met) in α-1-proteinase inhibitor (α-1-PI) to methionine sulfoxide (Met(O)) is known to cause depletion of its elastase inhibitory activity. To estimate the selectivity of different oxidants in converting Met to Met(O) in α-1-PI, we measured the molar ratio Met(O)/α-1-PI at total inactivation. This ratio was determined to be 1.2 for both the myeloperoxidase/H₂O₂/chloride system and the related compound NH₂Cl. With taurine monochloramine, another myeloperoxidase-related oxidant, 1.05 mol Met(O) were generated per mol α-1-PI during inactivation. These oxidants attack preferentially one Met residue in α-1-PI, which is identical with Met 358, as concluded from the parallelism of loss of elastase inhibitory activity and oxidation of Met. A similar high specificity for Met oxidation was determined for the xanthine oxidase-derived oxidants. In contrast, the ratio found for ozone and m-chloroperoxybenzoic acid was 6.0 and 5.0, respectively, indicating oxidation of additional Met residues besides the reactive site Met in α-1-PI, i.e. unselective action of these oxidants. Further studies were performed on the efficiency of oxidants for total depletion of the elastase inhibitory capacity of α-1-PI. Ozone and m-chloroperoxybenzoic acid were 10-fold less effective and the superoxide anion/hydroxyl radicals were 30–50-fold less effective to inactivate the elastase inhibitory activity as compared to the myeloperoxidase-derived oxidants. The myeloperoxidase-related oxidants are discussed as important regulators of α-1-PI activity in vivo.     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.     

Specific Labeling of the Phosphate Translocator in C(3) and C(4) Mesophyll Chloroplasts by Tritiated Dihydro-DIDS (1,2-Ditritio-1,2-[2,2' -Disulfo-4,4' -Diisothiocyano] Diphenylethane)

The phosphate translocator protein of C₃ and C₄ mesophyll chloroplast envelopes was specifically labeled using the anion exchange inhibitor, 1,2-ditritio-1,2-(2,2′ -disulfo-4,4′ -diisothiocyano) diphenylethane ([³H]₂-DIDS). Intact mesophyll chloroplasts were isolated from the C₃ plants, Spinacia oleracea L. (spinach) and Pisum sativum L. (pea), and the C₄ plant, Zea mays L. (corn). Chloroplasts were incubated with 5 to 50 μm [³H]₂-DIDS and, in addition, pea chloroplasts were also incubated with pyridoxal phosphate/tritiated sodium borohydride. The chloroplasts were washed, the envelopes isolated and solubilized. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, label from bound [³H]₂-DIDS was detected only in the 28- to 30-kilodalton protein (proposed C₃ phosphate translocator) for both C₃ and C₄ chloroplasts, as demonstrated by fluorography. In contrast, when pyridoxal phosphate/tritiated sodium borohydride was used to label pea chloroplasts, radioactivity was detected in several other bands in addition to the 29-kilodalton polypeptide. These findings suggest that DIDS is a much more specific inhibitor than reagents previously employed to study the phosphate translocator and could be used to isolate and characterize the differences in the C₃ and C₄ phosphate translocator protein(s).     

Pro-fluorescent probe with morpholine moiety and its reactivity towards selected biological oxidants

Biological oxidants participate in many processes in the human body. Their excessive production causes organelle damage, which may result in the accumulation of cytotoxic mediators and cell degradation and may manifest itself in various diseases. Peroxynitrite (ONOO⁻), hypochlorous acid (HOCl), hydrogen peroxide (H₂O₂), and peroxymonocarbonate (HOOCO₂⁻) are important oxidants in biology, toxicology, and various pathologies. Derivatives of coumarin, containing an oxidant-sensitive boronate group, have been recently developed for the fluorescent detection of inflammatory oxidants. Here, we report the synthesis and characterization of 4-[2-(morpholin-4-yl)-2-oxoethyl]-2-oxo-2H-chromen-7-yl boronic acid ( MpC-BA ) as a fluorescent probe for the detection of oxidants, with better solubility in water, high stability and fast response time toward peroxynitrite and hypochlorous acid. The effectiveness of the MpC-BA probe for the detection of peroxynitrite was measured by adding bolus ONOO⁻ or using the co-generating superoxide and nitrogen oxide system. MpC-BA is oxidized by ONOO⁻ to 7-hydroxy-4-[2-(morpholin-4-yl)-2-oxoethyl]-2H-chromen-2-one ( MpC-OH ). However, peroxynitrite-specific product ( MpC-H ) is formed in the minor reaction pathway. MpC-OH is also yielded in the reaction of MpC-BA with HOCl, and the subsequent formation of a chlorinated MpC-OH gives a specific product for HOCl ( MpC-OHCl ). H₂O₂ slowly oxidizes MpC-BA . However, the addition of NaHCO₃ increased the MpC-OH formation rate. We conclude that MpC-BA is potentially an improved fluorescent probe detecting peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of chlorinated and reduced coumarin(s) will help identify the oxidants detected.     

Phosphate Translocator of Mesophyll and Bundle Sheath Chloroplasts of a C(4) Plant, Panicum miliaceum L. : Identification and Kinetic Characterization

The phosphate translocator was identified in the envelope membranes of both mesophyll and bundle sheath chloroplasts of Panicum miliaceum L. by labeling with [1,2-³H]1,2-(2,2′ -disulfo-4,4′ -diisothiocyano)diphenylethane ([³H]H₂DIDS) and by using SDS-PAGE. Assay of ³²Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C₃ counterpart and can be regarded as C₄ types.     

Effect of osmotic stress on photosynthesis studied with the isolated spinach chloroplast : generation and use of reducing power

The effect of increasing assay medium sorbitol concentration from 0.33 to 1.0 molar on the photosynthetic reactions of intact and broken spinach (Spinacia oleracea L. var. Long Standing Bloomsdale) chloroplasts was investigated by monitoring O₂ evolution supported by the addition of glyceric acid 3-phosphate (PGA), oxaloacetic acid (OAA), 2,5-dimethyl-p-benzoquinone, and 2,6-dichlorophenolindophenol or as O₂ uptake with methyl viologen as acceptor.

Uncoupled 2,6-dichlorophenolindophenol-supported whole chain electron transport (photosystems I and II) was inhibited from the 0.33 molar rate by 14% and 48.6% at 0.67 and 1.0 molar sorbitol in the intact chloroplast and by only 0.4% and 25.0% in the broken chloroplast preparation. Whole chain electron flow from water to other oxidants (OAA, methyl viologen) was also inhibited at increased osmoticum in intact preparations while electron flow from water to methyl viologen, ferricyanide, and NADP in broken preparations did not demonstrate the osmotic response. Electron transport to 2,5-dimethyl-p-benzoquinone (photosystem II) from H₂O and to methyl viologen (photosystem I) from 3,3′-diaminobenzidine were found to be unaffected by osmolarity in both intact and broken preparations.

The stress response was more pronounced (26-38%) with PGA as substrate in the presence of 0.67 molar sorbitol than the inhibition found with uncoupled and coupled linear electron flow. In addition, substrate availability and ATP generated by cyclic photophosphorylation evaluated by addition of Antimycin A were found not to be mediating the full osmotic inhibition of PGA-supported O₂ evolution. In a reconstituted (thylakoids plus stromal protein) chloroplast system to which a substrate level of PGA was added, O₂ evolution was only slightly (7.8%) inhibited by increased osmolarity (0.33-0.67 molar sorbitol) indicating that the level of osmotic inhibition above that contributed by adverse effects on electron flow can be attributed to the functioning of the photosynthetic carbon reduction cycle within the intact chloroplasts.

     

Temperature and preillumination dependence of delayed fluorescence of spinach chloroplasts

Delayed fluorescence (luminescence) from spinach chloroplasts, induced by short saturating flashes, was studied in the temperature region between 0 and ?40 °C. At these temperatures, in contrast to what is observed at room temperature, luminescence at 40 ms after a flash was strongly dependent, with period four, on the number of preilluminating flashes (given at room temperature, before cooling). At ?35 °C luminescence of chloroplasts preilluminated with two flashes (the optimal preillumination) was about 15 times larger than that of dark-adapted chloroplasts. The intensity of luminescence obtained with preilluminated chloroplasts increased steeply below ?10 °C, presumably partly due to accumulation of reduced acceptor (Q^?), and reached a maximum at ?35 °C.In the presence of 50 mM NH₄Cl the temperature optimum was at ?15 °C; at this temperature luminescence was increased by NH₄Cl; at temperatures below ?20 °C luminescence at 40 ms was decreased by NH₄Cl. At room temperature a strongly enhanced 40-ms luminescence was observed after the third and following flashes. The results indicate that both the S₂ to S₃ and the S₃ to S₄ conversion are affected by NlH₄Cl.Inhibitors of Q^? reoxidation, like 3-(3, 4-dichlorophenyl)-1, 1- dimethylurea, did only slightly affect the preillumination dependence of luminescence at sub-zero temperatures if they were added after the preillumination. This indicates that these substances by themselves do not accelerate the deactivation of S₂ and S₃.     

The Coupling of Electron Flow to ATP Synthesis in Pea and Maize Mesophyll Chloroplasts : I. INTERACTION OF ADENINE NUCLEOTIDES AND ENERGY TRANSFER INHIBITORS WITH THE COUPLING FACTOR COMPLEX

The rate of nonphosphorylating electron transport (in the absence of ADP and inorganic phosphate) in well-coupled (ATP/2e⁻ = 0.9-1.1) maize mesophyll chloroplasts is not modulated by external pH (6.5-8.5), low levels of ADP or ATP, or energy transfer inhibitors, e.g. triphenyltin and Hg²⁺ ions. In contrast nonphosphorylating electron flow in pea chloroplasts is sensitive to alterations in medium pH, and to the presence of adenine nucleotides and energy transfer inhibitors in the assay medium. Although ATP is without effect on the rate of basal electron transport in maize chloroplasts, steady-state proton uptake is stimulated 3- to 5-fold by low levels of ATP. These results suggest that differences may exist in the manner in which the coupling factor complex controls proton efflux from the intrathylakoid space in C₃ and C₄ mesophyll chloroplasts.     

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     

Evidence for a light dependent increase of phosphoglucomutase activity in isolated, intact spinach chloroplasts

Phosphoglucomutase (PGM) activity was measured in spinach (Spinacia oleracea L.) chloroplasts. Initial enzyme activity in a chloroplast lysate was 5 to 10% of total activity measured with 1 micromolar glucose 1,6-bisphosphate (Glc 1,6-P₂) in the assay. Initial PGM activity increased 2- to 3-fold when chloroplasts were illuminated for 10 minutes prior to enzyme measurement and then decreased slowly in the dark. Measurements of total enzyme activity were unchanged by prior light treatment. Initial PGM activity from light treated chloroplasts was sufficient to account for in vivo rates of starch synthesis. Changes in PGM activity were affected by stromal pH and orthophosphate concentration. Photosynthetic inhibitors, dl-glyceraldehyde, glycolaldehyde, and glyoxylate, decreased and 3-phosphoglyceric acid increased light induced changes of PGM activity. Dark preincubation of chloroplasts with 10 millimolar dithiothreitol had no effect upon initial PGM activity, suggesting that light effects did not involve a sulfhydryl mechanism. Hexose monophosphate levels increased in illuminated chloroplasts. Activation of PGM in a chloroplast lysate by Glc 1,6-P₂ was maximal between pH 7.5 and 8.5. Stromal concentrations of Glc 1,6-P₂ were between 20 and 30 micromolar for both light and dark incubated chloroplasts and these levels should saturate PGM activity. Light dependent alterations of enzyme activity may be due to changes of phosphorylated PGM levels in the stroma or are the result of changes in residual activity by the dephosphorylated form of the enzyme. The above results indicate that PGM activity in spinach chloroplasts may be regulated by light, stromal pH, and Glc 1,6-P₂ concentration.     

Characterization of 4,4'-Diisothiocyano-2,2'-disulfonic Acid Stilbene Inhibition of 3-Phosphoglycerate-Dependent O(2) Evolution in Isolated Chloroplasts : Evidence for a Common Binding Site on the C(4) Phosphate Translocator for 3-Phosphoglycerate, Phosphoenolpyruvate, and Inorganic Phosphate

3-Phosphoglycerate (PGA)-dependent O₂ evolution by mesophyll chloroplasts of the C₄ plant, Digitaria sanguinalis L. Scop. (crabgrass), was inhibited by micromolar levels of 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS). As little as 1.8 micromolar DIDS added to the assay medium (containing 0.7 millimolar PGA) resulted in 80 to 100% inhibition of O₂ evolution. The extent of inhibition of O₂ evolution observed was dependent on various factors including: pH, concentration of DIDS to relative chlorophyll, concentration of PGA, and the time of addition of DIDS to the chloroplasts relative to addition of PGA.

Preincubation of crabgrass chloroplasts with micromolar levels of DIDS, followed by washing to remove any nonirreversibly bound DIDS, inhibited PGA-dependent O₂ evolution. Protection against this inhibition was afforded by preincubating the chloroplasts with various substrates before adding DIDS. For example, if the chloroplasts were first incubated with 8.3 millimolar PGA, phosphoenolpyruvate (PEP) or inorganic phosphate before adding 42 micromolar DIDS, the percentage of inhibition was decreased from 100% (without any substrate) to 0, 54, and 67%, respectively. 2-Phosphoglycerate caused a slight decrease in the inhibition (about 10%) and glucose-6-phosphate had no protective effect. If the chloroplasts were pretreated with DIDS initially, the inhibition could not be overcome by PGA, suggesting that DIDS acts as an irreversible inhibitor. Micromolar levels of DIDS also inhibited PGA dependent O₂ evolution by isolated chloroplasts of the C₃ plant barley. As with crabgrass, preincubation with PGA or inorganic phosphate resulted in a decrease in the DIDS inhibition, but PEP was very ineffective compared to the C₄ chloroplasts.

Oxalacetate-dependent O₂ evolution and its stimulation by the uncoupler, NH₄Cl, were unaffected by the addition of DIDS to crabgrass mesophyll chloroplasts. Furthermore, preincubation of the chloroplasts with DIDS (up to 65 micromolar) had no inhibitory effect on the extractable activity of NADP glyceraldehyde-3-P dehydrogenase and phosphoglycerate kinase. Inhibition by DIDS was interpreted to be at the substrate binding site of the phosphate translocator. The data further suggest that in C₄ crabgrass chloroplasts, PEP is transported on a carrier which also transports PGA.

     

Sulfate and sulfite translocation via the phosphate translocator of the inner envelope membrane of chloroplasts

The permeability of the inner envelope membranes of spinach (Spinacia oleracea) chloroplasts to sulfite and sulfate was investigated in vitro, using the technique of silicone oil centrifugal filtration. The results show that there is a permeability towards both ions, resulting in rates of uptake of about 1.0 (SO ₃ ^2- ) and 0.7 (SO ₄ ^2- ) mol mg chlorophyll^-1 h^-1 respectively (external concentration 2 mmol l^-1). The rates depend on the external concentration of the anions. Anion exchange experiments with ³⁵S-preloaded chloroplasts indicate that sulfite and sulfate are exchanged for inorganic phosphate, phosphoglyceric acid, and dihydroxyacetone phosphate with rates up to 14 nmol mg chlorophyll^-1 min^-1. There is no exchange for glucose-6-phosphate and malate. Because of the similarities to the transport of inorganic phosphate and triose phosphates the results give evidence that the phosphate translocator of the inner envelope membrane of chloroplasts is also involved in sulfite and sulfate transport — at least in part.Abbreviations DHAP dihydroxyacetone phosphate - PGA 3-phosphoglycerate - P_i inorganic phosphate - S_i sultite, sulfate     

Reactions between dimanganese,dirhenium, and manganese–rhenium decacarbonyl and oxidants

The dimetal decacarbonyls M₂(CO)₁₀, where M₂ = Mn₂, MnRe, and Re₂, do not react with anionic strong oxidants such as Fe(CN)₆³⁻ or IrCl₆²⁻ in CH₃CN solvent nor with neutral or cationic weak oxidants. Strong cationic oxidants such as NO⁺, Fe(phen)₃³⁺, and Cu²⁺ rapidly oxidize M₂(CO)₁₀ to 2M(CO)₅(NCCH₃)⁺ in acetonitrile solvent. The kinetics for the reaction suggest it proceeds according to a bimolecular outer sphere mechanism.     

Glutamine transport and the role of the glutamine translocator in chloroplasts

The transport of l-[¹⁴C]glutamine in oat (Avena sativa L.) and spinach (Spinacia oleracea L.) chloroplasts was studied by a conventional single-layer and a newly developed stable double-layer silicone oil filtering system. [¹⁴C]Glutamine was actively transported into oat chloroplasts against a concentration gradient. Metabolite uptake was greatly affected by the endogenous dicarboxylate pools, which could be easily changed by preloading the chloroplast with specific exogenous substrate. Glutamine uptake was decreased by 44 to 75% in oat chloroplasts preloaded with malate, 2-oxoglutarate (2-OG), and aspartate, but increased by 52% in chloroplasts preloaded with l-glutamate. On the other hand, the uptake of the other four dicarboxylates was decreased by 47 to 79% in chloroplasts preloaded with glutamine. In glutamine-preloaded chloroplasts the uptake of glutamine was inhibited only by l-glutamate. The observed inhibition by l-glutamate was competitive with an apparent K_i value of 32.1 millimolar in oat and 6.7 millimolar in spinach chloroplasts. This study indicates that there are two components involved in glutamine transport in chloroplasts. The major component was mediated via a specific glutamine translocator. It was specific for glutamine and did not transport other dicarboxylates except l-glutamate. A K_0.5 value of 1.25 millimolar and V_max of 45.5 micromoles per milligram of chlorophyll per hour were determined for the glutamine translocator in oat chloroplasts. The respective values were 1.0 millimolar and 16.7 micromoles per milligram of chlorophyll per hour in spinach chloroplasts. A three translocator model, involving the glutamine, dicarboxylate, and 2-OG translocators, is proposed for the reassimilation of photorespiratory NH₃ in chloroplasts of C₃ species. In this three-translocator model the additional transport of glutamine into the chloroplast is coupled to the export of glutamate via the glutamine translocator. This is an extension of the two-translocator model, involving the dicarboxylate and 2-OG translocators, proposed for spinach chloroplasts, (KC Woo, UI Flügge, HW Heldt 1987 Plant Physiol 84: 624-632).