Biomedical subjects

H W Heldt

Publications and source records attributed to H W Heldt.

At least 37 records · Page 2Linked to original sources

PubMed1991

Amino Acid and sucrose content determined in the cytosolic, chloroplastic, and vacuolar compartments and in the Phloem sap of spinach leaves.

Amino acid and sucrose contents were analyzed in the chloroplastic, cytosolic, and vacuolar compartments and in the phloem sap of illuminated spinach leaves (Spinacia oleracea L.). The determination of subcellular metabolite distribution was carried out by nonaqueous fractionation of frozen and lyophilized leaf material using a novel three-compartment calculation method. The phloem sap was collected by aphid stylets which had been severed by a laser beam. Subcellular analysis revealed that the amino acids found in leaves are located mainly in the chloroplast stroma and in the cytosol, the sum of their concentrations amounting to 151 and 121 millimolar, respectively, whereas the amino acid concentrations in the vacuole are one order of magnitude lower. The amino acid concentrations in the phloem sap are found to be not very different from the cytosolic concentrations, whereas the sieve tube concentration of sucrose is found to be one order of magnitude higher than in the cytosol. It is concluded that the phloem loading results in a preferential extraction of sucrose from the source cells.

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PubMed1990

On the regulation of spinach nitrate reductase.

A coupled assay has been worked out to study spinach (Spinacea oleracea L.) nitrate reductase under low, more physiological concentrations of NADH. In this assay the reduction of nitrate is coupled to the oxidation of malate catalyzed by spinach NAD-malate dehydrogenase. The use of this coupled system allows the assay of nitrate reductase activity at steady-state concentrations of NADH below micromolar. We have used this coupled assay to study the kinetic parameters of spinach nitrate reductase and to reinvestigate the putative regulatory role of adenine nucleotides, inorganic phosphate, amino acids, and calcium and calmodulin.

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PubMed1990

Involvement of na in active uptake of pyruvate in mesophyll chloroplasts of some c(4) plants : na/pyruvate cotransport.

An artificial Na(+) gradient across the envelope (Na(+) jump) enhanced pyruvate uptake in the dark into mesophyll chloroplasts of a C(4) plant, Panicum miliaceum (NAD-malic enzyme type) (J Ohnishi, R Kanai [1987] FEBS Lett 219:347). In the present study, (22)Na(+) and pyruvate uptake were examined in mesophyll chloroplasts of several species of C(4) plants. Enhancement of pyruvate uptake by a Na(+) jump in the dark was also seen in mesophyll chloroplasts of Urochloa panicoides and Panicum maximum (phosphoenolpyruvate carboxykinase types) but not in Zea mays or Sorghum bicolor (NADP-malic enzyme types). In mesophyll chloroplasts of P. miliaceum and P. maximum, pyruvate in turn enhanced Na(+) uptake in the dark when added together with Na(+). When flux of endogenous Na(+) was measured in these mesophyll chloroplasts preincubated with (22)Na(+), pyruvate addition induced Na(+) influx, and the extent of the pyruvate-induced Na(+) influx positively correlated with that of pyruvate uptake. A Na(+)/H(+) exchange ionophore, monensin, nullified all the above mutual effects of Na(+) and pyruvate in mesophyll chloroplasts of P. miliaceum, while it accelerated Na(+) uptake and increased equilibrium level of chloroplast (22)Na(+). Measurements of initial uptake rates of pyruvate and Na(+) gave a stoichiometry close to 1:1. These results point to Na(+)/pyruvate cotransport into mesophyll chloroplasts of some C(4) plants.

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PubMed1989

Effects of inorganic phosphate on the light dependent thylakoid energization of intact spinach chloroplasts.

The light dependent energization of the thylakoid membrane was analyzed in isolated intact spinach (Spinacia oleracea L.) chloroplasts incubated with different concentrations of inorganic phosphate (Pi). Two independent methods were used: (a) the accumulation of [(14)C]5,5-dimethyl-2,4-oxazolidinedione and [(14)C] methylamine; (b) the energy dependent chlorophyll fluorescence quenching. The inhibition of CO(2) fixation by superoptimal medium Pi or by adding glyceraldehyde-an inhibitor of the Calvin cycle-leads to an increased energization of the thylakoid membrane; however, the membrane energization decreases when chloroplasts are inhibited by suboptimal Pi. This specific ;low phosphate' effect could be partially reversed by adding oxaloacetate, which regenerates the electron acceptor NADP(+) and stimulates linear electron transport. The energization seen in low Pi is, however, always lower than in superoptimal Pi, even in the presence of oxaloacetate. Energization recovers in the presence of low amounts of N,N'-dicyclohexylcarbodiimide, which reacts with proton channels including the coupling factor 1 ATP synthase. N,N'-Dicyclohexylcarbodiimide has no effect on energization of chloroplasts in superoptimal Pi. These results suggest there is a specific ;low phosphate' proton leak in the thylakoids, and its origin is discussed.

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PubMed1989

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-(3)H]1,2-(2,2' -disulfo-4,4' -diisothiocyano)diphenylethane ([(3)H]H(2)DIDS) and by using SDS-PAGE. Assay of (32)Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C(3) counterpart and can be regarded as C(4) types.

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PubMed1988

Oxaloacetate and malate transport by plant mitochondria.

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10(-3). From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport.

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PubMed1988

Metabolite diffusion into bundle sheath cells from c(4) plants: relation to c(4) photosynthesis and plasmodesmatal function.

The present studies provide the first measurements of the resistance to diffusive flux of metabolites between mesophyll and bundle sheath cells of C(4) plants. Species examined were Panicum miliaceum, Urochloa panicoides, Atriplex spongiosa, and Zea mays. Diffusive flux of metabolites into isolated bundle sheath cells was monitored by following their metabolic transformation. Evidence was obtained that the observed rapid fluxes occurred via functional plasmodesmata. Diffusion constants were determined from the rate of transformation of limiting concentrations of metabolites via cytosolic enzymes with high potential velocities and favorable equilibrium constants. Values on a leaf chlorophyll basis ranged between 1 and 5 micromoles per minute per milligram of chlorophyll per millimolar gradient depending on the molecular weight of the metabolite and the source of bundle sheath cells. Diffusion of metabolites into these cells was unaffected by a wide variety of compounds including respiratory inhibitors, monovalent and divalent cations, and plant hormones, but it was interrupted by treatments inducing cell plasmolysis. The molecular weight exclusion limit for permeation of compounds into bundle sheath cells was in the range of 850 to 900. These cells provide an ideal system for the quantitative study of plasmodesmatal function.

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PubMed1987

The simultaneous determination of carbon dioxide release and oxygen uptake in suspensions of plant leaf mitochondria oxidizing glycine.

The construction and operation of a device for continuous measurement of CO(2) release by suspensions of respiring mitochondria is described. A combination of this device with a Clark-type O(2) electrode was used for simultaneous measurement of respiration and of CO(2) release by spinach and pea leaf mitochondria with glycine as substrate. Both mitochondrial preparations showed high rates of respiration and high respiratory control ratios. The addition of oxaloacetate not only inhibited O(2) uptake substantially, but also greatly stimulated glycine oxidation as monitored by CO(2) release. In spinach leaf mitochondria, the maximal rates of glycine oxidation thus obtained, were two times higher than the rate of glycine oxidation required at average rates of photorespiration. It is concluded from these results that under saturating conditions the capacity of glycine oxidation by intact mitochondria exceeds the capacity of glycine-dependent respiration.

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PubMed1987

Subcellular Metabolite Levels in Spinach Leaves : Regulation of Sucrose Synthesis during Diurnal Alterations in Photosynthetic Partitioning.

The alterations of subcellular metabolite levels during the day in spinach leaves have been investigated using nonaqueous density gradient centrifugation to separate chloroplasts, cytosol, and vacuole. The results provide direct evidence for the role of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase in regulating sucrose synthesis in leaves and also show that the phosphate translocator is kinetically limiting in vivo.

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PubMed1987

A Two-Translocator Model for the Transport of 2-Oxoglutarate and Glutamate in Chloroplasts during Ammonia Assimilation in the Light.

This study examines the transport of 2-oxoglutarate (2-OG) and other dicarboxylates during ammonia assimilation in illuminated spinach chloroplasts. The transport of all dicarboxylates examined was strongly inhibited by NH(4)Cl preincubation in the light. Treatment with NH(4)Cl caused a rapid depletion of the endogenous glutamate pool and a corresponding increase in endogenous glutamine content. The inhibition of transport activity by NH(4)Cl was apparently linked to its metabolism in the light because inhibition of glutamine synthetase activity by the addition of l-methionine sulfoximine or carbonylcyanide-m-chlorophenylhydrazone abolished this affect. Measurements of endogenous metabolite pools showed that malate was most rapidly exchanged during the uptake of all exogenous dicarboxylates examined. Depending on the exogenous substrates used, the apparent half-times of efflux measured for endogenous malate, aspartate and glutamate were 10, 10 to 30, and 15 to 240 seconds, respectively. The transport of 2-OG was also inhibited by malate. But chloroplasts preincubated with malate in the presence or absence of NH(4)Cl were found to have high transport activity similar to untreated chloroplasts. A two-translocator model is proposed to explain the stimulation of 2-OG transport as well as the stimulation of (NH(3), 2-OG)-dependent O(2) evolution by malate (KC Woo, CB Osmond 1982 Plant Physiol 69: 591-596) in isolated chloroplasts. In this model the transport of 2-OG on the 2-OG translocator and glutamate on the dicarboxylate translocator is coupled to malate counter-exchange in a cascade-like manner. This results in a net 2-OG/glutamate exchange with no net malate transport. Thus, during NH(3) assimilation the transport of 2-OG into and the export of glutamate out of the chloroplast occurs via the 2-OG and the dicarboxylate translocators, respectively.

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PubMed1987

N-ammonia assimilation, 2-oxoglutarate transport, and glutamate export in spinach chloroplasts in the presence of dicarboxylates in the light.

The direct incorporation of (15)NH(4)Cl into amino acids in illuminated spinach (Spinacia oleracea L.) chloroplasts in the presence of 2-oxoglutarate plus malate was determined. The amido-N of glutamine was the most highly labeled N-atom during (15)NH(4) assimilation in the presence of malate. In 4 minutes the (15)N-label of the amido-N of glutamine was 37% enriched. In contrast, values obtained for both the N-atom of glutamate and the amino-N of glutamine were only about 20% while that of the N-atom of aspartate was only 3%. The addition of malate during the assimilation of (15)NH(4)Cl and Na(15)NO(2) greatly increased the (15)N-label into glutamine but did not qualitatively change the order of the incorporation of (15)N-label into all the amino acids examined. This evidence indicates the direct involvement of the glutamine synthetase/glutamate synthase pathway for ammonia and nitrite assimilation in isolated chloroplasts. The addition of malate or succinate during ammonia assimilation also led to more than 3-fold increase in [(14)C]2-oxoglutarate transport into the chloroplast as well as an increase in the export of [(14)C]glutamate out of the chloroplast. Little [(14)C]glutamine was detected in the medium of the chloroplast preparations. The stimulation of (15)N-incorporation and [(14)C]glutamate export by malate could be directly attributed to the increase in 2-oxoglutarate transport activity (via the 2-oxoglutarate translocator) observed in the presence of exogenous malate.

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PubMed1986

Limitation of Photosynthesis by Carbon Metabolism : II. O(2)-Insensitive CO(2) Uptake Results from Limitation Of Triose Phosphate Utilization.

The occurrence of O(2)-insensitive photosynthesis at high quantum flux and moderate temperature in Spinacia oleracea was characterized by analytical gas exchange measurements on intact leaves. In addition photosynthetic metabolite pools were measured in leaves which had been rapidly frozen under defined gas conditions. Upon switching to low O(2) in O(2)-insensitive conditions the ATP/ADP ratio fell dramatically within one minute. The P-glycerate pool increased over the same time. Ribulose bisphosphate initially declined, then increased and exceeded the pool size measured in air. The pools of hexose monophosphates and UDPglucose were higher at a partial pressure of O(2) of 21 millibars than at 210 millibars. These results are consistent with the hypothesis that the rate of sucrose synthesis limited the overall rate of assimilation under O(2)-insensitive conditions.

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PubMed1985

Synthesis, storage, and stability of [4-14C]oxaloacetic acid.

A simple procedure for preparing [4-14C]oxaloacetic acid based on the reaction between [14C]HCO-3 and phosphoenolpyruvate catalyzed by phosphoenolpyruvate carboxylase is described. A simple method for preparing highly purified phosphoenolpyruvate carboxylase from maize leaves is described and the degradation of oxaloacetate under conditions of varying pH and divalent metal ion concentration is reported on. [4-14C]Oxaloacetic acid is stable for several months in 0.1 M HCl solution at -80 degrees C.

Carbon Radioisotopes↗

PubMed1985

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : V. Modulation of the Spinach Leaf Cytosolic Fructose 1,6-Bisphosphatase Activity in Vitro by Substrate, Products, pH, Magnesium, Fructose 2,6-Bisphosphate, Adenosine Monophosphate, and Dihydroxyacetone Phosphate.

How fructose 2,6-bisphosphate and metabolic intermediates interact to regulate the activity of the cytosolic fructose 1,6-bisphosphatase in vitro has been investigated. Mg(2+) is required as an activator. There is a wide pH optimum, especially at high Mg(2+). The substrate dependence is not markedly pH dependent. High concentrations of Mg(2+) and fructose 1,6-bisphosphate are inhibitory, especially at higher pH. Fructose 2,6-bisphosphate inhibits over a wide range of pH values. It acts by lowering the maximal activity and lowering the affinity for fructose 1,6-bisphosphate, for which sigmoidal saturation kinetics are induced, but the Mg(2+) dependence is not markedly altered. On its own, adenosine monophosphate inhibits competitively to Mg(2+) and noncompetitively to fructose 1,6-bisphosphate. In the presence of fructose 2,6-bisphosphate, adenosine monophosphate inhibits in a fructose 1,6-bisphosphate-dependent manner. In the presence of adenosine monophosphate, fructose 2,6-bisphosphate inhibits in Mg(2+)-dependent manner. Fructose 6-phosphate and phosphate both inhibit competitively to fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate does not affect the inhibition by phosphate, but weakens inhibition by fructose 6-phosphate. Dihydroxyacetone phosphate and hydroxypyruvate inhibit noncompetitively to fructose 1,6-bisphosphate and to Mg(2+), but both act as activators in the presence of fructose 2,6-bisphosphate by decreasing the S(0.5) for fructose 1,6-bisphosphate. A model is proposed to account for the interaction between these effectors.

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PubMed1985

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : VI. Regulation of the Cytosolic Fructose 1,6-Bisphosphatase in Spinach Leaves by an Interaction between Metabolic Intermediates and Fructose 2,6-Bisphosphate.

In this article, we propose a model describing how the spinach leaf cytosolic fructose 1,6-bisphosphatase is regulated in vivo by an interaction between fructose 2,6-bisphosphate and metabolic intermediates during photosynthesis. Previously published results are reanalyzed to provide a description of the way in which fluxes and metabolites vary in spinach leaves, depending on the relation between the supply of photosynthate and the demand for sucrose. The activity of the spinach leaf cytosolic fructose 1,6-bisphosphatase has been assayed in conditions simulating those expected in leaves as the rate of sucrose synthesis increases in response to rising rates of photosynthesis, and as sucrose synthesis declines in response to accumulation of sucrose in the leaf so that more starch is synthesized. The results show that regulation of the cytosolic fructose 1,6-bisphosphatase by alterations of fructose 2,6-bisphosphate, dihydroxyacetone phosphate, adenosine monophosphate, and phosphate can account for the alterations of flux found in vivo. The properties of a regulatory network, which allows the distribution of triose P between the Calvin cycle, sucrose synthesis, and starch synthesis to be balanced and adjusted, are described.

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PubMed1984

Measurement of subcellular metabolite levels in leaves by fractionation of freeze-stopped material in nonaqueous media.

This paper describes a technique for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the vacuole of spinach (Spinacia oleracea U.S.A. hybrid 424) leaves. Spinach leaves were freeze stopped and the frozen tissue was ground and lyophilized. The dry material was homogenized by sonication in a mixture of carbon tetrachloride and heptane, and fractionated by density gradient centrifugation. Measurements of the activity of marker enzymes in various subcellular compartments show the chloroplastic material mainly appearing in the lightest fractions and the cytosolic material in the middle of the gradient, whereas most of the vacuolar material is found in the heaviest fraction. Using the measured distributions of metabolites and of marker enzymes in each fraction of the gradient, the subcellular distribution of the metabolite can be calculated.As a first application, the new fractionation technique was used to investigate the subcellular contents of malate and sucrose in spinach leaves. The results show striking diurnal changes of sucrose and malate, with both substances primarily located in the vacuolar compartment. About three times more malate is present at the end of the day than at the end of the night. The sucrose content in the vacuole falls from a maximum of 45 millimolars at the end of the day to an almost undetectable value of approximately 1 millimolar at the end of the night.

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PubMed1984

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : I. Coordination of CO(2) Fixation and Sucrose Synthesis.

A mechanism is proposed for a feed-forward control of photosynthetic sucrose synthesis, which allows withdrawal of carbon from the chloroplast for sucrose synthesis to be coordinated with the rate of carbon fixation. (a) Decreasing the rate of photosynthesis of spinach (Spinacia oleracea, U.S. hybrid 424) leaf discs by limiting light intensities or CO(2) concentrations leads to a 2-to 4-fold increase in fructose 2,6-bisphosphate. (b) This increase can be accounted for by lower concentrations of metabolites which inhibit the synthesis of fructose 2,6-bisphosphate, such as dihydroxyacetone phosphate and 3-phosphoglycerate. (c) Thus, as photosynthesis decreases, lower levels of dihydroxyacetone phosphate should inhibit the cytosolic fructose bisphosphatase via simultaneously lowering the concentration of the substrate fructose 1,6-bisphosphate, and raising the concentration of the inhibitor fructose 2,6-bisphosphate.

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PubMed1984

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : II. Partitioning between Sucrose and Starch.

The role of fructose 2,6 bisphosphate in partitioning of photosynthate between sucrose and starch has been studied in spinach (Spinacia oleracea U.S. hybrid 424). Spinach leaf material was pretreated to alter the sucrose content, so that the rate of starch synthesis could be varied. The level of fructose 2,6-bisphosphate and other metabolites was then related to the accumulation of sucrose and the rate of starch synthesis. The results show that fructose 2,6-bisphosphate is involved in a sequence of events which provide a fine control of sucrose synthesis so that more photosynthate is diverted into starch in conditions when sucrose has accumulated to high levels in the leaf tissue. (a) As sucrose levels in the leaf rise, there is an accumulation of triose phosphates and hexose phosphates, implying an inhibition of sucrose phosphate synthase and cytosolic fructose 1,6-bisphosphatase. (b) In these conditions, fructose 2,6-bisphosphate increases. (c) The increased fructose 2,6-bisphosphate can be accounted for by the increased fructose 6-phosphate in the leaf. (d) Fructose 2,6-bisphosphate inhibits the cytosolic fructose 1,6-bisphosphatase so more photosynthate is retained in the chloroplast, and converted to starch.

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