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Biomedical subjects

G E Edwards

Publications and source records attributed to G E Edwards.

At least 73 records · Page 4Linked to original sources

Influence of Varying CO(2) and Orthophosphate Concentrations on Rates of Photosynthesis, and Synthesis of Glycolate and Dihydroxyacetone Phosphate by Wheat Chloroplasts.

Intact chloroplasts of wheat (Triticum aestivum) were isolated from mesophyll protoplasts. With decreasing concentrations of bicarbonate from 10 to 0.3 millimolar (pH 8.0), the optimal concentration of orthophosphate (Pi) for photosynthetic O(2) evolution decreased from a value of 0.1 to 0.2 millimolar to 0 to 0.025 millimolar. The extremely low Pi optimum for photosynthesis at the low bicarbonate levels of 0.3 millimolar was increased by lowering the O(2) concentration from 253 (21% gas phase) to 72 micromolar (6% gas phase). The relative amount of glycolate and dihydroxyacetone phosphate (DHAP) synthesized under high and low levels of bicarbonate and varying levels of Pi was determined. At low levels of bicarbonate, glycolate was the main product, whereas at high bicarbonate levels, DHAP was the main product. Most of the DHAP and glycolate was found in the extrachloroplastic fraction.The rate of photosynthesis at low levels of bicarbonate (0.3 millimolar) was as high as 75 to 95% of that at high levels of bicarbonate (10 millimolar) at the respective optimal levels of Pi. At low bicarbonate levels, and without Pi, there was little lag in photosynthetic O(2) evolution upon illumination in comparison to that of high bicarbonate levels and optimal levels of Pi. It is proposed that conditions which favor glycolate synthesis allow photosynthesis to continue without a depletion of internal Pi, whereas consumption of Pi may occur in the chloroplast during the net synthesis of organic phosphates under high levels of bicarbonate and without addition of Pi. At low bicarbonate levels, the extreme susceptibility of photosynthesis to inhibition by Pi may be due to excessive export of carbon from the chloroplast in the form of both glycolate and triose phosphate.

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Determination of NAD Malic Enzyme in Leaves of C(4) Plants : EFFECTS OF MALATE DEHYDROGENASE AND OTHER FACTORS.

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn(2+)-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.A model was developed which allowed the generation of theoretical curves describing the influence of malate dehydrogenase on the assay of malic enzyme activity. The experimental data obtained agreed closely with these curves. The conditions included 5 millimolar malate, 2 millimolar NAD, and 4 millimolar Mn(2+) (pH 7.2 to 7.8 at 30 degrees C). At low activities of malic enzyme (1 nanomole per minute per milliliter or less), the presence of malate dehydrogenase leads to a substantial overestimation of malic enzyme activity due to the Mn(2+)-catalyzed decarboxylation of oxaloacetate having a dominant effect. When the level of malic enzyme is greater than 1 nanomole per minute per milliliter, reversal of malate dehydrogenase has a dominant transient effect, causing a lag of up to several minutes, after which the change in absorbance reflects the true rate of malic enzyme. Independent of this effect is a lag in the activator-dependent rate, which could be eliminated by preincubating the enzyme with activator (coenzyme A).An assay procedure designed to minimize the influence of these effects is described. New data are presented on the activity of NAD malic enzyme in leaves of different subgroups of C(4) plants.

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Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : I. FACTORS AFFECTING NET CO(2) UPTAKE IN INTACT LEAVES AND CONTRIBUTION FROM RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE MEASURED IN VIVO AND IN VITRO.

As part of an extensive analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C(3) grass, we have examined the response of leaf gas exchange and ribulose-1,5-bisphosphate (RuBP) carboxylase activity to temperature. Emphasis was placed on elucidating the specific processes which regulate the temperature response pattern. The inhibitory effects of above-optimal temperatures on net CO(2) uptake were fully reversible up to 40 degrees C. Below 40 degrees C, temperature inhibition was primarily due to O(2) inhibition of photosynthesis, which reached a maximum of 65% at 45 degrees C. The response of stomatal conductance to temperature did not appear to have a significant role in determining the overall temperature response of photosynthesis. The intracellular conductance to CO(2) increased over the entire experimental temperature range, having a Q(10) of 1.2 to 1.4. Increases in the apparent Michaelis constant (K(c)) for RuBP carboxylase were observed in both in vitro and in vivo assays. The Q(10) values for the maximum velocity (V(max)) of CO(2) fixation by RuBP carboxylase in vivo was lower (1.3-1.6) than those calculated from in vitro assays (1.8-2.2). The results suggest that temperature-dependent changes in enzyme capacity may have a role in above-optimum temperature limitations below 40 degrees C. At leaf temperatures above 40 degrees C, decreases in photosynthetic capacity were partially dependent on temperature-induced irreversible reductions in the quantum yield for CO(2) uptake.

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Influence of Nitrate and Ammonia on Photosynthetic Characteristics and Leaf Anatomy of Moricandia arvensis.

The leaf anatomy and certain photosynthetic properties of nitrate- and ammonia-grown plants of Moricandia arvensis (L.) DC., a species previously reported to be a C(3)-C(4) intermediate, were investigated. Nitrate-grown plants had a high level of malate in the leaves while ammonia-grown plants had low levels of malate. In young leaves of nitrate-grown plants, there was a diurnal fluctuation of malate content, increasing during the day and decreasing during the night. Titratable acidity remained low in leaves of both nitrate- and ammonia-grown plants.In nitrate-grown plants, the activity of phosphoenolpyruvate (PEP) carboxylase was about 2-fold higher than in ammonia-grown plants, the latter having activity typical of C(3) species. Also, in nitrate-grown plants, the ratio of activities of ribulose 1,5-bisphosphate (RuBP) carboxylase/PEP carboxylase was lower than in ammonia-grown plants. Nitrate reductase activities were higher in nitrate- than in ammonia-grown plants and the greatest activity was found in younger leaves.With nitrate-grown plants, during a pulse-chase experiment the label in malate, as a percentage of the total labeled products, increased from about 7% after a 10-second pulse with (14)CO(2) up to 17% during a 5-minute chase with (12)CO(2). The pattern of (14)C labeling in various metabolites suggests the primary carboxylation is through RuBP carboxylase with a secondary carboxylation through PEP carboxylase. In similar experiments, with ammonia-grown plants, the percentage label in malate was only 0% to 4% with no increase in malate labeling during the chase period. The CO(2) compensation point was lower in nitrate-grown than ammonia-grown plants.There was no evidence of Kranz-like anatomy in either the nitrate or ammonia-grown plants. Mitochondria of bundle-sheath cells were strikingly positioned along the inner tangential wall. This might allow the chloroplasts of these cells to fix the mitochondrial photorespired CO(2) more effectively and contribute to the low CO(2) compensation point in the species. Chloroplasts of bundle-sheath cells and contiguous mesophyll cells were similar in size and structure in plants grown on different media, although chloroplast thylakoids and stromata of the ammonia-grown plants stained more intensely than those of nitrate-grown plants. In addition, irregular clusters of phytoferritin particles occurred in the chloroplasts of the ammonia-grown plants.The results indicate that the substantial activity of PEP carboxylase, incorporation of CO(2) into malate, the high malate content, and in part the relatively low CO(2) compensation point in Moricandia arvensis may be accounted for by metabolism of nitrate rather than by a state of C(3)-C(4) intermediacy.

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Isolation and purification of intact peroxisomes from green leaf tissue.

Intact peroxisomes were prepared from green leaves of a number of C(3) species, both monocots and dicots. A protoplast extract from which chloroplasts have been removed by a 1-minute 10,000g centrifugation was applied to a step gradient of 5, 15, 30, and 45% Percoll containing 0.5 molar sucrose, 0.1% BSA, and 25 millimolar Hepes-KOH (pH 7.5). After centrifugation, a peroxisomal fraction with low contamination by chloroplastic and mitochondrial markers was recovered from the 30/45% Percoll interface. This fraction was passed through a Sepharose 2B column to remove the Percoll which resulted in a peroxisomal preparation exhibiting high intactness (estimated from enzyme latency) and stability.

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Inhibition of photosynthetic carbon metabolism in isolated chloroplasts by iodoacetol phosphate.

Carbon dioxide-dependent and 3-phosphoglycerate (PGA)-dependent O(2) evolution by isolated chloroplasts of wheat is inhibited by micromolar levels of iodoacetol phosphate (IAP). Loss of the activity is time-dependent and a higher concentration of PGA increases the half-time for inhibition (e.g. at 40 micromolar IAP the half-time is about 0.5 minutes at 1 millimolar PGA compared to 1.5 minutes at 10 millimolar PGA). A marked inhibition of NADP glyceraldehyde-3-P dehydrogenase was observed when chloroplasts were pretreated with micromolar levels of IAP, osmotically shocked, and several stromal enzymes assayed.Extraction of enzymes from wheat protoplasts and treatment with IAP showed that nanomolar levels of the compound completely inhibited NAD and NADP glyceraldehyde-3-P dehydrogenase and the half-time for inactivation at 5 nanomolar IAP was about 1 minute. The inhibitory effect of IAP was not reversed by passing the enzyme extract through a column of Sephadex G-25. The concentration of IAP required for inhibition of the chloroplastic triose phosphate isomerase is about three orders of magnitude higher than that with glyceraldehyde-3-P dehydrogenase. Micromolar levels of IAP had no effect on ribulose-1,5-bisphosphate carboxylase. Inhibition of chloroplast photosynthesis and of glyceraldehyde-3-P dehydrogenase in protoplast extracts with IAP follows pseudo first-order kinetics.Pretreatment of chloroplasts with IAP did not inactivate the phosphate translocator of the chloroplast envelope. Iodoacetol phosphate may enter the chloroplasts through the phosphate translocator since a high concentration of IAP (0.5 millimolar) competitively inhibits uptake of (32)Pi. Iodoacetol phosphate had no effect on ferricyanide-dependent O(2) evolution with isolated chloroplasts. Also, IAP had no effect on light-dependent fixation of CO(2) through the carboxylation phase of the C(4) pathway with protoplast extracts of crabgrass mesophyll cells. The site of IAP inhibition of photosynthesis with wheat chloroplasts is suggested to be through the inactivation of glyceraldehyde-3-P dehydrogenase.

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Nocturnal Accumulation of Malic Acid Occurs in Mesophyll Tissue without Proton Transport to Epidermal Tissue in the Inducible Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum: EVIDENCE AGAINST A PREVIOUS HYPOTHESIS.

The inducible Crassulacean acid metabolism plant, Mesembryanthemum crystallinum, accumulates malic acid, i.e. equivalent amounts of malate anions and protons in the mesophyll cells at night. Levels of malate and titratable acidity are low in the epidermal tissue and do not change significantly during the day/night cycle. This result is in contrast to a recent report (Bloom 1979 Plant Physiol 64: 919-923) that the synthesis of malic acid during dark CO(2) fixation is associated with an equivalent exchange of inorganic cations from epidermal tissue with protons in the mesophyll cells.

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Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS.

Potato tuber mitochondria oxidized exogenous NADH and exogenous NADPH at similar rates; the electron transfer inhibitor rotenone did not inhibit the oxidation of either substrate. Submitochondrial particles, prepared from potato tuber mitochondria, exhibited a greater capacity to oxidize NADH than NADPH; rotenone inhibited the oxidation of NADH by 29% and the oxidation of NADPH by 16%. The oxidation of both NADH and NADPH by potato mitochondria exhibited pH optima of 6.8, and although substantial NADH oxidase activity was observed at pH 8.0, little NADPH oxidase activity was detected at that pH. The oxidation of NADPH by the mitochondria was more sensitive to inhibition by EDTA than was the oxidation of NADH.The sulfhydryl reagents N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuriphenyl sulfonic acid, and mersalyl inhibited the oxidation of exogenous NADPH by the mitochondria whereas NADH oxidation was unaffected at similar concentrations of inhibitor. The data suggest that exogenous NADPH is oxidized by potato mitochondria via a dehydrogenase primarily situated on the outer face of the inner mitochondrial membrane that is neither the dehydrogenase involved with endogenous NADH oxidation nor with exogenous NADH oxidation.

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Localization of glycerate kinase and some enzymes for sucrose synthesis in c(3) and c(4) plants.

The localization of some key enzymes leading to sucrose synthesis in photosynthetic tissue of C(3) and C(4) species was investigated. These included UDP-glucose (UDPG) pyrophosphorylase, sucrose phosphate synthetase, and glycerate kinase. Whether glycerate kinase is localized exclusively in the chloroplast or partly outside the chloroplast could influence the fate of carbon flow to sucrose through the glycolate pathway.In the C(3) species wheat, intact chloroplasts were isolated from protoplasts. Following separation of the chloroplasts by differential centrifugation and by sucrose density gradient centrifugation, UDPG pyrophosphorylase, sucrose phosphate synthetase, and sucrose synthetase were found outside the chloroplast while glycerate kinase was localized in the chloroplast.In the C(4) species (maize, NADP-malic enzyme-type; Panicum miliaceum, NAD-malic enzyme type and Brachiaria erucaeformis, phosphoenolpyruvate carboxykinase type) the distribution of UDPG pyrophosphorylase and glycerate kinase between mesophyll and bundle sheath cells and their intracellular localization in mesophyll protoplasts was determined. Substantial activity of UDPG pyrophosphorylase was found in both mesophyll and bundle sheath cells while glycerate kinase was localized only in mesophyll cells. From C(4) mesophyll protoplasts, UDPG pyrophosphorylase was found to be cytoplasmic while glycerate kinase appears exclusively localized in the chloroplasts as determined by differential centrifugation and sucrose density gradients.It seems that in both C(3) and C(4) plants, the terminal steps of sucrose synthesis occur exclusively in the cytoplasm while carbon flow in the glycolate pathway to sucrose must occur through glycerate kinase in the chloroplasts. The localization of glycerate kinase in mesophyll cells of C(4) plants may have further implications for intercellular flow of carbon during metabolism in the glycolate pathway in these species.

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Photosynthesis in Isolated Chloroplasts of the Crassulacean Acid Metabolism Plant Sedum praealtum.

Intact chloroplasts were isolated from protoplasts of the Crassulacean acid metabolism plant Sedum praealtum D.C. Typical rates of CO(2) fixation or CO(2)-dependent O(2) evolution ranged from 20 to 30 micromoles per milligram chlorophyll per hour and could be stimulated 30 to 50% by several Calvin cycle intermediates. The pH optimum for CO(2) fixation was 7.0 to 7.6 with considerable activity as low as pH 6.4. Low concentrations of orthophosphate (Pi) (optimum 0.4 millimolar) stimulated photosynthesis while high concentrations (5 millimolar) caused some inhibition. Both CO(2) fixation and CO(2)-dependent O(2) evolution exhibited a relatively long lag phase (4 to 6 minutes) which remained constant between 0.4 to 5 millimolar Pi. The lag phase could be decreased by addition of dihydroxyacetone-phosphate or ribose 5-phosphate. Further results are presented which suggest these chloroplasts have a functional phosphate translocator.A long lag period at optimum Pi concentration, no effect of high Pi levels on the lag phase, lack of severe inhibition of photosynthesis by high Pi concentrations, and the relatively low pH optimum for photosynthesis are characteristics unlike those previously reported with chloroplasts of C(3) plants.

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Quantum Requirement for Photosynthesis in Sedum praealtum during Two Phases of Crassulacean Acid Metabolism.

The quantum requirement (QR) for photosynthesis in Sedum praealtum, a Crassulacean acid metabolism plant, was compared with that of wheat, a C(3) plant, and maize, a C(4) plant, at 30 C. During the deacidification phase in S. praealtum, approximately 16 moles quanta were absorbed per mole malate consumed. This is equivalent to 16 moles quanta per mole CO(2) fixed, assuming 1 mole CO(2) is assimilated per mole malate decarboxylated. This QR for Crassulacean acid metabolism is similar to that of the C(3) or C(4) plant under atmospheric conditions, even though there are considerable differences in the biochemistry of photosynthesis. During late-afternoon C(3)-like fixation of atmospheric CO(2) in S. praealtum, the QR was relatively high with values of 41 under 21% O(2) and 19 under 2% O(2). During the deacidification phase in S. praealtum, the relatively low QR can be accounted for by the repression of photorespiration and saturation of photosynthesis from the elevated CO(2) concentration in the leaves during malate decarboxylation.

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Intracellular Localization of Some Key Enzymes of Crassulacean Acid Metabolism in Sedum praealtum.

The intracellular locations of six key enzymes of Crassulacean acid metabolism were determined using enzymically isolated mesophyll protoplasts of Sedum praealtum D.C. Data from isopycnic sucrose density gradient centrifugation established the chloroplastic location of pyruvate Pi dikinase, the mitochondrial location of NAD-linked malic enzyme, and exclusively nonparticulate (not associated with chloroplasts, peroxisomes, or mitochondria) locations of phosphoenolpyruvate carboxylase, NADP-linked malic enzyme, enolase, and phosphoglycerate mutase. The consequences of this enzyme distribution with respect to compartmentalization of the pathway and the transport of metabolites in Crassulacean acid metabolism are discussed.

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Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum.

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C(4) plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O(2) evolution by chloroplasts isolated from protoplasts.Chloroplasts were isolated from protoplasts by several passages of the protoplasts through a 20-micrometer nylon mesh. Mesophyll chloroplasts were judged approximately 90 to 95% intact and bundle sheath chloroplasts 80 to 90% intact based on retention of chloroplast marker enzymes and the ferricyanide test for intactness. It was necessary to include 10 millimolar MgCl(2) in media for osmotically shocking the chloroplasts in order to obtain maximum and linear rates of ferricyanide-dependent O(2) evolution.Chloroplasts isolated from mesophyll protoplast preparations had low rates of light-dependent O(2) evolution in the presence of 10 millimolar NaHCO(3) (0.13 micromoles per milligram chlorophyll per minute) in comparison to bundle sheath chloroplasts (1 to 2.5 micromoles per milligram chlorophyll per minute). The mesophyll chloroplasts catalyze high rates of 3-phosphoglycerate-dependent O(2) evolution (2 to 4 micromoles per milligram chlorophyll per minute). Orthophosphate but not phosphoenolpyruvate inhibited the 3-phosphoglycerate-dependent O(2) evolution by the mesophyll chloroplasts. Rates of O(2) evolution were much higher with mesophyll than with bundle sheath chloroplasts in the presence of pyruvate plus oxaloacetate. The results are discussed in relation to the proposed function of these chloroplasts during C(4) photosynthesis.

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Isolation and Oxidative Properties of Intact Mitochondria from the Leaves of Sedum praealtum: A Crassulacean Acid Metabolism Plant.

A procedure is described for preparing intact mitochondria from leaves of Sedum praealtum D.C., a plant showing Crassulacean acid metabolism. These mitochondria oxidized malate, pyruvate, alpha-ketoglutarate, succinate, NADH, NADPH, and isocitrate with good respiratory control and ADP/O ratios better than those observed in mitochondria from other photosynthetic tissues.Malate oxidation was very resistant to inhibition by rotenone. Glycine oxidation was very slow with poor respiratory control and was resistant to rotenone inhibition. Antimycin A completely inhibited the oxidation of both NADH and NADPH. The oxidation of isocitrate, malate, succinate, and alpha-ketoglutarate was partially inhibited by antimycin A and cyanide. Overall rates of substrate oxidation were slow on a protein basis, but purification of the mitochondrial preparations on a linear sucrose gradient removed a large amount of nonmitochondrial protein. The original mitochondrial preparations contained little glycolate oxidase activity, and most of this activity was removed by the sucrose gradient.

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Oxidation of reduced nicotinamide adenine dinucleotide phosphate by plant mitochondria.

Mitochondria isolated from various plant tissues (leaves, etiolated shoots and hypocotyls, and stem tubers) oxidize exogenous NADPH with respiratory control values and ADP:O ratios similar to those obtained with exogenous NADH as substrate. In all the mitochondria investigated, the electron-transfer inhibitors rotenone and amytal each had the same effect on the oxidation of NADPH as they had on the oxidation of NADH. The oxidation of exogenous NADPH by white potato tuber mitochondria was much more sensitive to inhibition by citrate or ethylene glycol bis-(beta-aminoethyl ether)-N,N-tetraacetic acid than was the oxidation of NADH. Mitochondria isolated from aged beetroot slices showed an increased capacity for the oxidation of exogenous NADH (compared with mitochondria from fresh tissue) but no such increase in the capacity to oxidize exogenous NADPH. These results suggest that exogenous NADPH and NADH are oxidized via different flavoproteins in plant mitochondria.

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