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H W Heldt

Publications and source records attributed to H W Heldt.

At least 73 records · Page 4Linked to original sources
PubMed1978

Subcellular metabolite concentrations. Dependence of mitochondrial and cytosolic ATP systems on the metabolic state of perfused rat liver.

Mitochondrial and cytosolic contents of adenine nucleotides and phosphate were measured in perfused rat livers employing a technique of fractionation of freeze-fixated tissue in non-aqueous solvents. From the subcellular contents the mitochondrial and cytosolic concentrations of ATP, ADP, AMP and phosphate and the phosphorylation potentials of the subcellular ATP systems were calculated. An attempt was made to elucidate the relationship between mitochondrial and cytosolic adenine nucleotide systems and the dependency on the metabolic state of the liver. The following results were obtained: 1. Under all metabolic conditions studied the mitochondrial ATP/ADP ratios were considerably lower than the cytosolic ratios (mitochondria: 0.1-0.7; cytosol: 2-11). 2. The ATP/ADP ratios calculated from overall tissue contents reflect mainly the cytosolic ratios. 3. An inverse relationship was found between mitochondrial and cytosolic ATP/ADP ratios, i.e. when the mitochondrial ratios tended to increase, the cytosolic ratios decreased and vice versa. 4. The phosphorylation potentials calculated from the subcellular concentrations were higher in the cytosol than in the mitochondria. The potential difference varied between 11 and 3 kj/mol in livers from fed and starved rats, respectively. 5. In the presence of mitochondrial inhibitors, i.e. amytal, dinitrophenol and carboxyatractyloside, the potential difference between the subcellular ATP systems decreased predominantly due to an increase in the mitochondrial ATP/ADP ratios. 6. A correlation between mitochondrial ATP/ADP ratios and the respiratory rates was not observed, but the subcellular ratios appeared to correlate with the rate of glycolysis. When the rate of lactate + pyruvate production was increased, the cytosolic ATP/ADP ratios were increased, too, whereas the mitochondrial ratios tended to decrease. 7. The adenine nucleotides in the cytosol appear to be in near equilibrium catalysed by the adenylate kinase. In the mitochondria, the AMP concentration is much lower than to be expected under equilibrium conditions. These results were discussed with respect to rate control of processes involved in ATP generation, i.e. oxidative phosphorylation, adenine nucleotide translocation and glycolysis.

Adenosine Diphosphate↗
PubMed1978

Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts.

The uptake of phosphate and phosphorylated compounds into the chloroplast stroma has been studied by silicone layer filtering centrifugation. 1. Inorganic phosphate, 3-phosphoglycerate, dihydroxyacetone phosphate and glyceraldehyde phosphate are transported across the envelope leading to an accumulation in the chloroplast stroma. This uptake proceeds by a counter exchange with phosphate and phosphorylated compounds present there. 2. The transport shows saturation characteristics allowing the determination of Km and V. 3. The phosphorylated compounds transported act as competitive inhibitors of the transport. From measurements of the Km and Ki the specificity of the transport is described. 4. The transport of inorganic phosphate and 3-phosphoglycerate is inhibited by p-chloromercuriphenyl sulfonate, pyridoxal 5'-phosphate and trinitrobenzene sulfonate. 5. The activation energy of phosphate transport as determined from the temperature dependence is evaluated to be 16 kcal (0--12 degrees C). 6. It is concluded that inorganic phosphate, 3-phosphoglycerate, dihydroxy-acetone phosphate and glyceraldehyde phosphate are transported by the same carrier, which has been nominated phosphate translocator. 7. Simultaneous measurements of the proton concentration in the medium and the transport into the chloroplasts show that the transfer of 3-phosphoglycerate involves a transfer of a proton into the same direction. 8. Measurements of the pH dependence of the transport indicate that all substances including 3-phosphoglycerate are transported by the phosphate translocator as divalent anions. 9. The physiological function of the phosphate translocator is discussed.

Biological Transport↗
PubMed1978

The mechanism of the control of carbon fixation by the pH in the chloroplast stroma. Studies with nitrite-mediated proton transfer across the envelope.

1. CO2 fixation of intact spinach chloroplasts is inhibited by nitrite in a pH-dependent mode. At pH 7.3 in the medium 1 mM NaNO2 and at pH 7.9 5 mM NaNO2 were required for 50% inhibition. 2. The addition of nitrite leads to an acidificiation in the stroma. It appears that nitrite renders the envelope permeable for protons resulting in a breakdown of the pH gradient between the external space and the stroma. 3. In view of earlier results on the pH sensitivity of C02 fixation it is concluded that this pH shift in the stroma is responsible for the observed inhibition of CO2 fixation by nitrite. 4. Octanoate and to some extent also high concentrations of bicarbonate and acetate have a similar effect as nitrite in inhibiting CO2 fixation through an acidification in the stroma. 5. The levels of the intermediates of the CO2 fixation cycle were measured. A strong rise of the levels of fructose- and sedoheptulose biphosphates and a concomitant decrease of the corresponding monophosphates was observed during inhibition of CO2 fixation. It appears that the enzymatic steps of the CO2 fixation cycle responsible for the overall inhibition of CO2 fixation caused by lowering of the H+ concentration in the stroma are fructose- and sedopheptulose bisphosphatase. These two enzymes have an important function in the light regulation of CO2 fixation.

Ammonium Chloride↗
PubMed1978

Dicarboxylate transport across the inner membrane of the chloroplast envelope.

The uptake of radioactively labeled dicarboxylates into the sorbitol-impermeable 3H2O space (the space surrounded by the inner envelope membrane) of spinach chloroplasts has been studied by means of silicone layer filtering centrifugation. 1. Malate, aspartate and a number of other dicarboxylates are rapidly transported across the envelope leading to an accumulation in the chloroplasts. This uptake proceeds mainly by a counterexchange with the dicarboxylates present there. 2. The dicarboxylate transport shows saturation characteristics allowing the determination of Km and V. 3. All dicarboxylates transported act as competitive inhibitors of the transport. 4. The activation energy of the transport as determined from the temperature dependency is evaluated to be 7 kcal/mol. 5. The rate of dicarboxylate transport is influenced by illumination, the countertransported molecules and the pH in the medium. These changes effect the transport velocity, whereas the corresponding Km values are not altered. 6. It is discussed whether there is more than one carrier involved in dicarboxylate transport in spinach chloroplasts.

Biological Transport↗
PubMed1977

The distribution of metabolites between spinach chloroplasts and medium during photosynthesis in vitro.

1. The formation of metabolites in the stroma compartment of isolated chloroplasts during carbon fixation, and their export to the medium, have been investigated using improved techniques. 2. Rapid separation of photosynthesising chloroplasts from the medium, accompanied by simultaneous quenching of metabolism was achieved by using silicone oil layer filtering centrifugation under illumination. Metabolites were separated by microscale ion-exchange chromatography. Quantitative determination of each metabolite was based on labelling with 32P. 3. It was found that fixed carbon was exported from the chloroplasts only as triose phosphate and phosphoglycerate, and to a minor extent, as pentose monophosphate. The main compounds accumulating in the stroma were hexose and heptose monophosphates and phosphoglycerate. A marked decrease in the concentration of inorganic phosphate in the stroma during the first 5 min of illumination was accompanied by a complementary increase in organic phosphate so that the total amount of phosphate within the chloroplasts remained constant. 4. The concentration difference for phosphoglycerate between the stroma and the medium was much higher than for triose phosphate or inorganic phosphate, although all three compounds are transported across the inner membrane of the chloroplast envelope by the same carrier. It was concluded that the efflux of phosphoglycerate was restricted.

Adenosine Triphosphate↗
PubMed1977

Transport of hexoses across the liver-cell membrane.

The uptake of radioactively labelled hexoses into the cellular space of isolated liver cells has been studied using silicone layer filtering centrifugation. From the hexoses tested, D-glucose is transported most rapidly followed by D-galactose and D-fructose. The rate of L-glucose transport is only 5% of that of D-glucose. This transport enables the concentration of free aldohexoses in the cellular space to reach the external concentration. For D-glucose the rate of transport into the cell largely exceeds the rate of metabolic conversion. This is different with D-fructose where the rate of transport is considerably lower but the rate of metabolism higher. Here the transport may even be a limiting step. The transport of D-glucose and D-galactose shows a saturation characteristic, whereas D-fructose appears not to be saturatable. The Km for D-glucose is found to be 30 mM. All these hexoses compete with each other for transportation. The temperature dependency of D-glucose reveals an activation energy of 22 kcal (92 kJ)/mol (4-18 degrees C) and 7 kcal (29 kJ)/mol (18-37 degrees C). The transport of D-glucose, D-galactose and D-fructose in inhibited by cytochalasin B, phloretin and phlorizin. It is not dependent on Na+ ions. Preliminary results showed no stimulation of the transport by insulin. It is concluded that D-glucose and other hexoses are transported by carrier-mediated diffusion across the plasma membrane of liver cells. This transport shows a large resemblance to the transport of D-glucose into human erythrocytes.

Animals↗
PubMed1977

Role of orthophosphate and other factors in the regulation of starch formation in leaves and isolated chloroplasts.

Starch synthesis in leaves was increased by phosphate starvation or by treatments which decreased cytoplasmic orthophosphate levels (such as mannose feeding). Usually less than 30% of the total carbon fixed during CO(2) assimilation was incorporated into starch in spinach (Spinacia oleracea L.), spinach beet (Beta vulgaris), and tobacco (Nicotiana tabacum) leaves.In isolated spinach chloroplasts, formation of starch from CO(2) was usually less than in leaves. In the absence of significant levels of 3-phosphoglycerate, concentrations of phosphate as low as 1 mm (in the medium) or 10 mm (in the stroma) almost completely inhibited starch synthesis. The inhibitory action of phosphate could be overcome by 3-phosphoglycerate. The controlling factor of starch synthesis appeared to be the ratio of phosphoglycerate to orthophosphate rather than the stromal hexose monophosphate concentration, and it is suggested that this control is exerted via the phosphate translocator and the known allosteric regulation of ADP-glucose pyrophosphorylase. Starch synthesis was also favored by the presence of dihydroxyacetone phosphate and by high light and high temperature. Oxygen was inhibitory, probably owing to carbon drain into glycolate. Starch formation by intact chloroplasts could not be promoted by added glucose or glucose 6-phosphate.Starch mobilization in the dark was promoted by orthophosphate and phosphate-dependent mobilization was inhibited by phosphoglycerate. The principal products of starch breakdown in the presence of phosphate were the transport metabolites dihydroxyacetone phosphate and 3-phosphoglycerate. Formation of these compounds from starch was stimulated by ATP or oxaloacetate. In a phosphate-independent reaction, starch was also converted to neutral products such as maltose and glucose. The rates of phosphate-dependent starch degradation phosphorolysis were very much higher than those of starch hydrolysis for which there was no phosphate requirement.

Journal Article↗
PubMed1976

Light-dependent changes of the Mg2+ concentration in the stroma in relation to the Mg2+ dependency of CO2 fixation in intact chloroplasts.

(1) Light-dependent changes of the Mg2+ content of thylakoid membranes were measured at pH 8.0 and compared with earlier measurements at pH 6.6. In a NaCl and KCl medium, the light-dependent decrease in the Mg2+ content of the thylakoid membranes at pH 8.0 is found to be 23 nmol Mg2+ per mg chlorophyll, whereas in a sorbitol medium it is 83 nmol Mg2+ per mg chlorophyll. (2) A light dependent increase in the Mg2+ content of the stroma was detected wjem chloroplasts were subjected to osmotic shock, amounting to 26 nmol/mg chlorophyll. Furthermore, a rapid and reversible light-dependent efflux of Mg2+ has been observed in intact chloroplasts when the divalent cation ionophore A 23 187 was added, indicating a light-dependent transfer of about 60 nmol of Mg2+ per mg chlorophyll from the thylakoid membranes to the stroma. (3) CO2 fixation, but not phosphoglycerate reduction, could be completely inhibited when A 23 187 was added to intact chloroplasts in the absence of external Mg2+. If Mg2+ was then added to the medium, CO2 fixation was restored. Half of the maximal restoration was achieved with about 0.2 mM Mg2+, which is calculated to reflect a Mg2+ concentration in the stroma of 1.2 mM. The further addition of Ca2+ strongly inhibits CO2 fixation. (4) The results suggest that illumination of intact chloroplasts causes an increase in the Mg2+ concentration of 1-3 mM in the stroma. Compared to the total Mg2+ content of chloroplasts, this increase is very low, but it appears to be high enough to have a possible function in the light regulation of CO2 fixation.

Calcimycin↗
PubMed1976

Transfer of substrates across the chloroplast envelope.

The chloroplast represents a relatively autonomous metabolic compartment within the plant cell. It is surrounded by an envelope consisting of two membranes of which the inner membrane is the functional barrier. Utilizing the energy of light the chloroplast is able to synthesize dihydroxyacetonephosphate from carbon dioxide and water. To provide the cell with this substrate, inorganic phosphate is required. In the case of phosphate deficiency the product of CO2 fixation may be temporarily stored within the chloroplast as starch. Specific transport processes across the inner envelope membrane permit the transfer of metabolites between the chloroplast and the cytosol. The phosphate translocator facilitates the export of dihydroxyacetone phosphate in exchange for inorganic phosphate. It also catalyzes a shuttle for inorganic phosphate with 3-phosphoglycerate, permitting the indirect transfer of reducing equivalents and of ATP from the chloroplast to the cytosol. The dicarboxylate carrier transporting various dicarboxylates may be suited for the transfer of reducing equivalents from the cytosol into the chloroplast. The ATP translocator, catalyzing a transport of ATP into the chloroplast in exchange for ADP, appears to be important for providing the chloroplast with ATP during the night phase, as required for the mobilization of starch.

Biological Transport↗
PubMed1975

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark.

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 degrees C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3.Phosphoglycerate reduction, which is a partial reaction of CO2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO2 fixation without affecting phosphoglycerate reduction. This inhibition of CO2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO2 fixation in the light. This demonstrates that CO2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.

Adenosine Triphosphate↗