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H W Heldt

Publications and source records attributed to H W Heldt.

At least 55 records · Page 3Linked to original sources
PubMed1984

Control of Photosynthetic Sucrose Synthesis by Fructose 2,6-Bisphosphate : III. Properties of the Cytosolic Fructose 1,6-Bisphosphatase.

The cytosolic fructose 1,6-bisphosphatase from spinach (Spinacia oleracea U.S. hybrid 424) leaves has been partially purified and its response to fructose 2,6-bisphosphate, AMP, and fructose 1,6-bisphosphate studied, using concentrations present in the cytosol during photosynthesis. In the presence of fructose 2,6-bisphosphate, the substrate saturation kinetics for fructose 1,6-bisphosphate are sigmoidal, with half-maximal activity being attained in 0.1 to 1 millimolar concentration range. The inhibition is enhanced by AMP. Using these results, and information published elsewhere on metabolite concentrations, it is discussed how fructose 1,6-bisphosphatase activity will vary in vivo in response to alterations in the availability of triose phosphate and AMP, and the accumulation of the product, fructose 6-phosphate.

Journal Article↗
PubMed1983

Regulation of Sucrose Synthesis by Cytoplasmic Fructosebisphosphatase and Sucrose Phosphate Synthase during Photosynthesis in Varying Light and Carbon Dioxide.

The aim of this work was to investigate whether sucrose synthesis in the cytosol of leaf cells is regulated in response to the supply of energy and organic carbon from the chloroplast. Fluxes into sucrose and metabolite levels in wheat (Triticum aestivum var Timmo) leaf protoplasts were compared in a range of light intensities and CO(2) concentrations, showing that sucrose-phosphate synthase and the cytosolic fructose-1,6-bisphosphatase are inhibited in situ when the supply of trioseP from the chloroplasts decreases. Such a regulation might aid CO(2) fixation in limiting conditions by permitting stromal metabolites to be maintained at higher levels than would otherwise be possible.

Journal Article↗
PubMed1983

A role for fructose 2,6-bisphosphate in the regulation of sucrose synthesis in spinach leaves.

The subcellular distribution of fructose 2,6-bisphosphate in spinach (Spinacia oleracea) leaves was studied using nonaqueous fractionation, showing that all, or almost all, is located in the cytosol. The amount of fructose 2,6-bisphosphate present in leaves during the diurnal cycle was measured and compared to the accumulation of starch and sucrose, and the amounts of selected phosphorylated intermediates in the leaf. Upon illumination, the level of fructose 2,6-bisphosphate decreases, but prolonged illumination leads to an increase in the level to above that found in the dark, which accompanies the onset of rapid accumulation of starch in the leaf.

Journal Article↗
PubMed1982

On the Participation of Phosphoribulokinase in the Light Regulation of CO(2) Fixation.

CO(2) fixation by a suspension of isolated spinach chloroplasts was terminated by turning off the light, and changes of metabolite levels in the chloroplast stroma and the surrounding medium were assayed. Whereas CO(2) fixation comes to a total stop within 15 seconds, a conversion of triose phosphates to heptose, hexose, and pentose monophosphates is found to occur for 1 to 2 minutes afterwards. It seems from these data that an inactivation of fructose and sedoheptulose bisphosphatases proceeds with a lag period. In contrast, the conversion of pentose monophosphates to ribulose 1,5-bisphosphate is inhibited immediately after the stop of illumination. As the stromal level of freely available ATP was not depleted under this condition, these data demonstrate that ribulose 5-phosphate kinase was very rapidly inactivated after darkening of the chloroplasts. Essentially, the same effect is also observed when CO(2) fixation is partially inhibited by addition of moderate concentrations of m-chlorocarbonyl phenylhydrazone, partially uncoupling photophosphorylation. It appears from these results, that the activity of ribulose 5-phosphate kinase is not only regulated by light through the mediation of reduced carriers like thioredoxin but also by alternative parameters, e.g. stromal metabolite levels.

Journal Article↗
PubMed1982

Rapid fractionation of wheat leaf protoplasts using membrane filtration : the determination of metabolite levels in the chloroplasts, cytosol, and mitochondria.

A technique is presented for measuring the in vivo metabolite levels in the chloroplast stroma, the cytosol, and the mitochondrial matrix of wheat (Triticum aestivum, var ;Timmo') leaf protoplasts, in which membrane filtration is used to prepare fractions enriched in the different subcellular fractions within 0.1 seconds after disruption of the protoplasts. By closing a syringe, protoplasts are forced through a net and disrupted, diluting the cytosol into the medium and also releasing intact chloroplasts and mitochondria which can then be immediately removed on membrane filters placed behind the nylon net. By varying the membrane filters, different filtrates are obtained corresponding to (a) mainly cytosol, or (b) cytosol and mitochondria with only low levels of chloroplasts; alternatively, (c) the entire protoplast contents are obtained by omitting the filters. The filtrates are immediately split, half flowing into HClO(4) where they are immediately quenched for subsequent metabolite analyses; the other half flows into detergent and is used to monitor the exact distribution of marker enzymes in each individual fractionation. Using the measured distributions of metabolite and of marker enzymes in the three filtrates, the subcellular distribution of the metabolite can be algebraically calculated. The method is presented using ATP as an example.The quench time (0.1 second) made possible by membrane filtration is considerably faster than has been possible in the previously developed techniques using silicone oil centrifugation for chloroplasts (1 second) or mitochondria (1 minute). This rapid quench makes it possible to investigate subcellular pools which have a rapid turnover, like the adenine nucleotides.

Journal Article↗
PubMed1982

Adenine nucleotide levels in the cytosol, chloroplasts, and mitochondria of wheat leaf protoplasts.

Recently, a new method has been described, in which membrane filtration is used to allow the levels of adenine nucleotides in the chloroplast stroma, the cytosol, and the mitochondrial matrix to be measured. This method is now used to investigate the effect of illumination, of respiratory inhibitors, and of uncouplers on the distribution of ATP, ADP, and AMP in wheat (Triticum aestivum var. ;Timmo') leaf protoplasts. (a) The adenine nucleotides are apparently equilibrated by adenylate kinase in the stroma and the cytosol, but not in the mitochondrial matrix. (b) The ATP/ADP quotient in the cytosol is considerably higher than that in the mitochondrial matrix or the chloroplast stroma. (c) A large gradient exists between the ATP/ADP quotients in the cytosol and the mitochondrial matrix in the dark, with a very low ATP/ADP quotient in the mitochondria. This gradient is lowered by uncouplers or respiratory inhibitors showing that, as in animal tissues, it reflects the energization of the mitochondria. (d) In the dark, the stromal ATP/ADP is lower than in the light, and appears to be maintained, at least in part, by import from the cytosol. (e) The cytosolic ATP/ADP, however, actually decreases in the light. This contradicts the widespread assumption, that export of photosynthetically produced ATP from the chloroplast leads to an increase in the cytosolic ATP/ADP, which then inhibits oxidative phosphorylation in the mitochondria. (f) The mitochondrial ATP/ADP increases in the light, and the gradient between the cytosol and mitochondrial matrix falls. This is also difficult to understand in terms of an inhibition of oxidative phosphorylation in the light due to a lack of ADP in the cytosol. (g) The significance of the measured variations in the adenine nucleotide pools are discussed with respect to the diurnal carbohydrate metabolism in a leaf, and to the metabolic function of the chloroplast, the cytosol and the mitochondria.

Journal Article↗
PubMed1981

Physiological rates of starch breakdown in isolated intact spinach chloroplasts.

Starch breakdown with rates above 10 muatom carbon per mg chlorophyll per hour has been monitored in spinach chloroplasts and compares favorably with the rates in whole leaves. Intact starch-loaded chloroplasts were prepared from protoplasts to avoid rupture during mechanical homogenization and rapid centrifugation. Particular attention was paid to the identification of all the products of starch degradation and to measuring the actual rates of their accumulation. The products of starch breakdown included triose phosphate, 3-phosphoglycerate, CO(2), glucose, and some maltose. Comparison of the rates of metabolism of added glucose and of the conversion of starch to phosphorylated intermediates showed that starch phosphorolysis was the major pathway leading to phosphorylated endproducts. From the results, the relative contribution of phosphorolysis and hydrolysis to starch breakdown and the contribution of glycolysis and the oxidative pentose phosphate cycle can be estimated. Phosphate has a large influence on the metabolism of the chloroplast in the dark.

Journal Article↗
PubMed1981

Changes in the subcellular distribution of metabolites due to ethanol oxidation in the perfused rat liver.

The subcellular distribution of metabolites involved in the transfer of reducing equivalents across the mitochondrial membrane was studied in perfused livers from fed rats. A tenfold increase in the flux rate of the malate-aspartate shuttle and the inhibition of the citrate cycle due to ethanol oxidation, were reflected by characteristic changes in the cytosolic and mitochondrial concentrations of malate, 2-oxoglutarate, aspartate, glutamate and citrate. The data suggest that the malate-aspartate shuttle is triggered by a decrease in the cytosolic oxaloacetate concentration which, due to the cytosolic aspartate aminotransferase equilibrium, leads to an increased efflux of 2-oxoglutarate and aspartate from the mitochondria in exchange for malate and glutamate, respectively. The first site at which the citrate cycle is inhibited appears to be the level of 2-oxoglutarate oxidation.

Animals↗
PubMed1980

Metabolite levels during induction in the chloroplast and extrachloroplast compartments of spinach protoplasts.

The light activation of photosynthesis has been investigated in spinach palisade cell protoplasts. (1) After a short induction period, maximal rates of photosynthesis are achieved. (2) [14C]Bicarbonate initially labels anionic compounds in the chloroplast and then in the extrachloroplast compartments. These pools saturate within 2-4 min and radioactivity accumulates mainly in sucrose in the extrachloroplast compartment, in starch and in cationic compounds. (3) Enzymic determinations were made of metabolite levels during the induction period in the chloroplast and extrachloroplast compartments. There is no general build-up of intermediates. Perturbations of individual intermediates occurred, consistent with the activation of specific enzymes. (4) It is suggested that fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase may limit flux in the Calvin cycle during induction. (5) The onset of sucrose synthesis is not accompanied by accumulation of intermediates in the cytosol. It is suggested that sucrose phosphate synthase or sucrose phosphate phosphatase is activated. (6) Measurements of metabolites in whole leaves during a 24 h illumination cycle confirmed that substrates are not depleted during the dark period, and that the onset of photosynthesis is not accompanied by a rise in intermediate levels. (7) It is concluded that the causes of the induction lag in protoplasts can differ from those in isolated chloroplasts.

Chloroplasts↗
PubMed1980

Energy charge, phosphorylation potential and proton motive force in chloroplasts.

Adenylate concentrations were measured in intact chloroplasts under a variety of conditions. Energy charge was significant in the dark and increased in the light, but remained far below values expected from observed phosphorylation potentials in broken chloroplasts, which were 80 000 M-1 or more in the light. With nitrite as electron acceptor, phosphorylation potentials in intact chloroplasts were about 80 M-1 in the dark and only 300 M-1 in the light. Similar phosphorylation potentials were observed, when oxaloacetate, phosphoglycerate or bicarbonate were used as substrates. delta G'ATP was -42 kJ/mol in darkened intact chloroplasts, -46 kJ/mol in illuminated intact chloroplasts and -60 kJ/mol in illuminated broken chloroplasts. Uncoupling by NH4Cl, which stimulated electron transport to nitrite or oxaloacetate and decreased the proton gradient, failed to decrease the phosphorylation potential of intact chloroplasts. Also, it did not increase the quantum requirement of CO2 reduction. It is concluded that the proton motive force as conventionally measured and phosphorylation potentials are far from equilibrium in intact chloroplasts. The insensitivity of CO2 reduction and of the phosphorylation potential to a decrease in the proton motive force suggests that intact chloroplasts are over-energized even under low intensity illumination. However, such a conclusion is at variance with available data on the magnitude of the proton motive force.

Adenosine Diphosphate↗
PubMed1980

Balance between Metabolite Accumulation and Transport in Relation to Photosynthesis by Isolated Spinach Chloroplasts.

The relationship between the rate of orthophosphate (Pi) transport into the stroma and the rate of CO(2) fixation by intact chloroplasts was investigated. High Pi concentrations in the medium lead to a depletion of stromal metabolites, due to excessive Pi transport into the stroma, resulting in the inhibition of CO(2) fixation. This inhibitory effect of Pi is released by inhibitors of Pi transport, such as pyrophosphate, citrate or pyridoxal-5-phosphate. The latter compound appeared to be specially valuable in inhibiting Pi transport without affecting stromal reactions.The Pi optima of CO(2) fixation were studied when the rate of CO(2) fixation or the rate of Pi transport was varied by the application of specific inhibitors. For optimal performance the rates of CO(2) fixation and Pi transport have to be matched in such a way, that transport neither limits nor exceeds CO(2) fixation. This accounts for the large variation in the Pi optima for CO(2) fixation by different chloroplast preparations.

Journal Article↗
PubMed1980

Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts.

A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross-contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1,5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis.

Journal Article↗
PubMed1980

Subcellular distribution of di- and tricarboxylates and pH gradients in perfused rat liver.

The subcellular distribution of malate, 2-oxoglutarate, citrate, aspartate and glutamate and the mitochondrial and cytosolic pH values were determined in perfused livers from fed and fasted rats. The method of fractionation of the freeze-dried tissue in non-aqueous solvents was employed. The following results were obtained: 1) Di- and tricarboxylates are not equally distributed between mitochondria and cytosol. Under the two metabolic conditions studied, the concentrations of citrate and 2-oxoglutarate are higher and those of glutamate and aspartate are lower in the mitochondria than in the cytosol. The distribution of malate varies with the metabolic state. 2) From the distribution of 5,5-dimethyl-2,4-oxazolidinedione it was calculated that the mitochondrial matrix has a more alkaline milieu than the cytosol. The cytosol is also more acidic than the perfusate. The pH difference between mitochondria and cytosol is 0.3 in the fed state and 0.6 in the fasted one, whereas the pH difference between perfusate and cytosol is 0.4 in the fed state and 0.2 in the fasted one. 3) In livers from fed rats, citrate, malate and 2-oxoglutarate appear to be distributed according to the pH gradient across the mitochondrial membrane, whereas in the fasted state, the distribution does not correspond to the pH gradient. Glutamate and aspartate do not follow the pH gradient in either metabolic state. 4) The data indicate that the pH gradient across the mitochondrial membrane does not reflect the membrane potential or the energy state of mitochondria in the intact cell.

Animals↗