scholarly journals Purification of highly intact plastids from various heterotrophic plant tissues: analysis of enzymic equipment and precursor dependency for starch biosynthesis

1993 ◽  
Vol 296 (2) ◽  
pp. 395-401 ◽  
Author(s):  
H E Neuhaus ◽  
O Batz ◽  
E Thom ◽  
R Scheibe
Keyword(s):  
Starch Synthesis ◽  
Dihydroxyacetone Phosphate ◽  
Low Degree ◽  
Fructose 1,6 Bisphosphatase ◽  
Percoll Gradients ◽  
Phosphoglyceric Acid ◽  
Barley Leaves ◽  
Centrifugation Step ◽  
Cellular Components ◽  
Hexose Phosphates

Starting with a protocol originally developed for the purification of intact plastids from cauliflower buds [Journet and Douce (1985) Plant Physiol. 79, 458-467] we have modified this method to obtain intact heterotrophic plastids from etiolated barley leaves (Hordeum vulgare) and pea (Pisum sativum) and maize (Zea mays) endosperm. Two subsequent centrifugation steps on Percoll gradients were performed, the first as an isopycnic, the second as zonal, centrifugation step in a swing-out rotor. Percoll density and centrifugation time were adjusted for the various tissues. The obtained plastid preparations are characterized by a low degree of contamination with other cellular components and an intactness of at least 90%. In isolated maize endosperm amyloplasts, starch synthesis is driven by exogenously applied hexose phosphates (glucose 6-phosphate and glucose 1-phosphate) rather than by dihydroxyacetone phosphate. The hexose-phosphate-dependent starch synthesis is strictly dependent upon the intactness of the plastids and is increased up to 9-fold when ATP and 3-phosphoglyceric acid are added to the incubation medium. The occurrence of fructose-1,6-bisphosphatase and malate dehydrogenases in some plastid types is discussed in relation to their possible role in starch synthesis.

scholarly journals ADP-glucose drives starch synthesis in isolated maize endosperm amyloplasts: characterization of starch synthesis and transport properties across the amyloplast envelope

1997 ◽  
Vol 324 (2) ◽  
pp. 503-509 ◽  
Author(s):  
Torsten MÖHLMANN ◽  
Joachim TJADEN ◽  
Gundrun HENRICHS ◽  
Paul W. QUICK ◽  
Rainer HÄUSLER ◽  
...  
Keyword(s):  
Starch Synthesis ◽  
Dihydroxyacetone Phosphate ◽  
Maize Endosperm ◽  
Endosperm Tissue ◽  
Plastid Envelope ◽  
Phosphorylated Compounds ◽  
Saturation Kinetics ◽  
Uptake Data ◽  
Hexose Phosphates

We recently developed a method of purifying amyloplasts from developing maize (Zea mays L.) endosperm tissue [Neuhaus, Thom, Batz and Scheibe (1993) Biochem. J. 296, 395–401]. In the present paper we analyse how glucose 6-phosphate (Glc6P) and other phosphorylated compounds enter the plastid compartment. Using a proteoliposome system in which the plastid envelope membrane proteins are functionally reconstituted, we demonstrate that this type of plastid is able to transport [14C]Glc6P or [32P]Pi in counter exchange with Pi, Glc6P, dihydroxyacetone phosphate and phosphoenolpyruvate. Glucose 1-phosphate, fructose 6-phosphate and ribose 5-phosphate do not act as substrates for counter exchange. Besides hexose phosphates, ADP-glucose (ADPGlc) also acts as a substrate for starch synthesis in isolated maize endosperm amyloplasts. This process exhibits saturation kinetics with increasing concentrations of exogenously supplied [14C]ADPGlc, reaching a maximum at 2 mM. Ultrasonication of isolated amyloplasts greatly reduces the rate of ADPGlc-dependent starch synthesis, indicating that the process is dependent on the intactness of the organelles. The plastid ATP/ADP transporter is not responsible for ADPGlc uptake. Data are presented that indicate that ADPGlc is transported by another translocator in counter exchange with AMP. To analyse the physiology of starch synthesis in more detail, we examined how Glc6P- and ADPGlc-dependent starch synthesis in isolated maize endosperm amyloplasts interact. Glc6P-dependent starch synthesis is not inhibited by increasing concentrations of ADPGlc. In contrast, the rate of ADPGlc-dependent starch synthesis is reduced by increasing concentrations of ATP necessary for Glc6P-dependent starch synthesis. The possible modes of inhibition of ADPGlc-dependent starch synthesis by ATP are discussed with respect to the stromal generation of AMP required for ADPGlc uptake.

Verteilung und Wanderung von Phosphoglycerat zwischen den Chloroplasten und dem Cytoplasma während der Photosynthese

1965 ◽  
Vol 20 (9) ◽  
pp. 890-898 ◽  
Author(s):  
W. Urbach ◽  
M. A. Hudson ◽  
W. Ullrich ◽  
K. A. Santarius ◽  
U. Heber
Keyword(s):  
Turnover Rate ◽  
Rapid Decrease ◽  
Permeability Barrier ◽  
Dihydroxyacetone Phosphate ◽  
Cyanide Poisoning ◽  
Similar Extent ◽  
Slow Increase ◽  
Phosphoglyceric Acid ◽  
Inhibition Of Metabolism ◽  
Isolated Chloroplasts

The distribution of phosphoglyceric acid (PGA) * between chloroplasts and cytoplasm of leaf cells during transients from dark to light and vice versa has been investigated. The data indicate that pools of PGA in the chloroplasts and cytoplasm are interchangeable and that PGA may function as a transport metabolite in actively metabolising leaf cells. These views are supported by the following results:1. In the presence of 14CO2 and light, labelled PGA rapidly appears in the cytoplasm, even though the carboxydismutase reaction, in which 14C enters into PGA, proceeds in the chloroplasts.2. After less than 1 min. illumination in the presence of 14CO2, the distribution of labelled PGA between chloroplasts and cytoplasm reaches an equilibrium, which is then maintained. The same distribution is to be found by enzymatic analyses of the total pools of PGA in chloroplasts and cytoplasm. When equilibrium is reached, the percentages of both 14C labelled and of total PGA to be found in the chloroplasts of Spinach and Elodea are approximately 75% and 35 —40% respectively.3. In both the chloroplasts and the cytoplasm, the levels of PGA first decrease after illumination to a fraction of the original dark levels and then show a concomitant slow increase. On darkening a further very rapid increase in PGA occurs in chloroplasts and cytoplasm.4. In photosynthetically active leaf material the rate of decrease in the level of cytoplasmic PGA, as observed after 12 —15 secs. illumination, is higher than the turnover rate of PGA in respiration.5. Upon illumination, aqueously isolated chloroplasts, suspended in isotonic sucrose buffer, reduce added PGA to dihydroxyacetone phosphate and other products far faster than they reduce added NADP. Whereas PGA reduction is not increased by ultrasonic disintegration of the chloroplasts, the reduction of NADP is stimulated. This indicates that whereas the movement of NADP is prevented by a permeability barrier, the transferance of PGA across the chloroplast membrane occurs easily.6. In illuminated Elodea shoots the inhibition of metabolism by cyanide after 15 secs. photosynthesis in the presence of Η14CO3⊖ leads to a rapid decrease in PGA. This applies to both the 14C labelled PGA and the total PGA to a similar extent. The decrease in PGA amounts from 70 — 85% of the original dark levels. Since the chloroplasts of Elodea contain only 35—40% of the total PGA of the cell, a fall in the level of PGA as a result of the cyanide poisoning obviously occurs not only in the chloroplasts, but also in the cytoplasm. Since cyanide effectively inhibits cytochrome oxidase, while PGA reduction in the chloroplasts is relatively resistant, the large decrease in PGA suggests that part of the cytoplasmic PGA is transferred into the chloroplasts and reduced there.

Salt Tolerance and Regulation of Enzymes of Starch Synthesis in Cassava (Manihot esculenta Crantz)

1982 ◽  
Vol 9 (5) ◽  
pp. 509 ◽  
Author(s):  
JS Hawker ◽  
GM Smith
Keyword(s):  
Manihot Esculenta ◽  
Starch Synthesis ◽  
Starch Synthase ◽  
Manihot Esculenta Crantz ◽  
Adpglucose Pyrophosphorylase ◽  
Storage Organs ◽  
Porous Growth ◽  
Phosphoglyceric Acid ◽  
And Storage ◽  
Nutrient Solutions

The growth rate of cassava plants (Manihot esculenta cv. MAUS7) decreased with increasing concentrations of NaCl from 0 to 75 mM in nutrient solutions supplied regularly in a porous growth medium in a glasshouse. Tuber weight was reduced to one-half between 30 and 50 mM NaCl and there was some burning of apical leaves at 50 and 75 mM NaCl. By comparison with other plants, this cultivar of cassava can be considered to have medium sensitivity to salinity. Na+ and Cl- concentrations increased in all tissues with increasing concentrations of supplied NaCl, except that Na+ remained low in laminae until the 75 mM treatment. K+ levels decreased in tubers. Starch concentrations remained the same in tubers, and K+ stimulated starch bound ADPglucose starch synthase by 1 .5-fold. Leaves and tubers contained activities of ADPglucose pyrophosphorylase and ADPglucose-starch synthase similar to those found in leaves and storage organs of other starch synthesizing plants. ADPglucose pyrophosphorylase from leaves was stimulated 20-fold by 3-phosphoglyceric acid (3PGA) while the enzyme from tubers was almost completely dependent on 3PGA at pH 8.5. The A0.5 values for 3PGA (the concentration required for one-half maximal activation) for the leaf and tuber enzymes at pH 8.5 were 1.31 mM and 7.41 mM respectively. At pH 7.5 the leaf enzyme was stimulated 26-fold and the tuber enzyme was again almost completely dependent on 3PGA. The A0.5 values at pH 7.5 were 1.17 mM and 3.8 mM, respectively. The I0.5 values for PI (concentrations required to cause 50% inhibition) in the presence of 3PGA were 2 mM, 0.25 mM and 0.04 mM for leaf enzyme at pH 8.5 and tuber enzyme at pH 8.5 and 7.7 respectively. The results support the view that it is not possible to generalize about the magnitude of the control of ADPglucose synthesis in leaves as opposed to non-chlorophyllous tissues.

scholarly journals Reconstitution of the hexose phosphate translocator from the envelope membranes of wheat endosperm amyloplasts

1996 ◽  
Vol 319 (3) ◽  
pp. 717-723 ◽  
Author(s):  
Ian J TETLOW ◽  
Caroline G BOWSHER ◽  
Michael J EMES
Keyword(s):  
Inorganic Phosphate ◽  
Competitive Inhibitor ◽  
Dihydroxyacetone Phosphate ◽  
Carrier Protein ◽  
Sugar Phosphate ◽  
Wheat Endosperm ◽  
Single Carrier ◽  
Phosphate Translocator ◽  
Envelope Membranes ◽  
Hexose Phosphates

Amyloplasts were isolated and purified from wheat endosperm and the envelope membranes reconstituted into liposomes. Envelope membranes were solubilized in n-octyl β-D-glucopyranoside and mixed with liposomes supplemented with 5.6 mol% cholesterol to produce proteoliposomes of defined size, which showed negligible leakage of internal substrates. Transport experiments with proteoliposomes revealed a counter-exchange of glucose 1-phosphate (Glc1P), glucose 6-phosphate (Glc6P), inorganic phosphate (Pi), 3-phosphoglycerate and dihydroxyacetone phosphate. The Glc1P/Pi counter-exchange reaction exhibited an apparent Km for Glc1P of 0.4 mM. Glc6P was a competitive inhibitor of Glc1P transport (Ki 0.8 mM), and the two hexose phosphates could exchange with each other, indicating the operation of a single carrier protein. Glc1P/Pi antiport in proteoliposomes showed an exchange stoichiometry at pH 8.0 of 1 mol of phosphate transported per mol of sugar phosphate.

Properties of Phosphoenolpyruvate Carboxykinase Operative in C4 Pathway Photosynthesis

1977 ◽  
Vol 4 (2) ◽  
pp. 207 ◽  
Author(s):  
MD Hatch ◽  
S Mau
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Reverse Direction ◽  
Nucleoside Triphosphate ◽  
Dihydroxyacetone Phosphate ◽  
C4 Pathway ◽  
Phosphoglyceric Acid ◽  
Michaelis Constants ◽  
Chloris Gayana ◽  
Fructose 1,6 Bisphosphate ◽  
Chloris Gayana Kunth

A procedure is described for partially purifying phosphoenolpyruvate carboxykinase [ATP : oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from leaves of Chloris gayana Kunth. In three steps the enzyme was purified about 60-fold with 22% recovery of activity. This procedure removes enzymes, particularly malate dehydrogenase, that preclude the use of a simple spectrophotometric assay for phosphoenolpyruvate carboxykinase. The activity of the enzyme in the direction of oxaloacetate decarboxylation was about 10 times that in the reverse direction. At the optimal pH of 8.0, ATP was the preferred nucleoside triphosphate but CTP, UTP, GTP and ITP were also active. A requirement for Mn2+ could not be replaced by Mg2+. The Michaelis constants for oxaloacetate and ATP were 0.035 mM and 0.024 nM, respectively. The photosynthetic intermediates fructose 1,6-bisphosphate, 3-phosphoglyceric acid and dihydroxyacetone phosphate significantly inhibited the enzyme at concentrations in the region of 1-5 mM. Unlike the phosphoenolpyruvate carboxykinase from other sources, the capacity of the leaf enzyme to catalyse the decarboxylation of oxaloacetate to pyruvate was negligible. The properties of the enzyme are discussed in relation to its proposed role in C4 pathway photosynthesis.

scholarly journals Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway

1996 ◽  
Vol 315 (2) ◽  
pp. 455-460 ◽  
Author(s):  
Eduardo KESSI ◽  
Victoria GUIXÉ ◽  
Ana PRELLER ◽  
URETA URETA
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Dihydroxyacetone Phosphate ◽  
Indirect Pathway ◽  
Ic50 Value ◽  
Glucose Incorporation ◽  
Relationship Of ◽  
Hexose Phosphates

Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor–product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H,U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but significant and reproducible detritiation (13%) in glycogen was observed. Co-injection of [U-14C]glucose and 3-mercaptopicolinate resulted in marked inhibition of glycogen labelling. Half-maximal inhibition was observed at 0.58 mM 3-mercaptopicolinate, which agrees with the IC50 value (0.47 mM) for the inhibition in vitro of phosphoenolpyruvate carboxykinase activity. We conclude that in frog oocytes most of the glucosyl units are incorporated into glycogen by an indirect pathway involving breakdown of glucose to lactate, which is then converted into glycogen via gluconeogenesis. Both processes, glycolytic degradation of glucose to lactate and subsequent reconversion of the latter into hexose phosphates and glycogen, occur in the same cell.

scholarly journals Regulatory Properties of Apple Leaf Cytosolic Fructose-1,6-bisphosphatase

HortScience ◽  
2004 ◽  
Vol 39 (4) ◽  
pp. 761C-761
Author(s):  
Rui Zhou* ◽  
Lailiang Cheng
Keyword(s):  
Enzyme Activity ◽  
Adenosine Monophosphate ◽  
Competitive Inhibitor ◽  
Inhibitory Effect ◽  
Carbon Partitioning ◽  
Dihydroxyacetone Phosphate ◽  
Saturation Curve ◽  
Fruit Species ◽  
Apparent Homogeneity ◽  
Fructose 1,6 Bisphosphatase

Cytosolic fructose-1,6-bisphosphatase (cytoFBPase) (EC 3.1.3.11) occupies a strategic site in sucrose synthesis and has been demonstrated to play a key role in carbon partitioning between sucrose and starch in non-sorbitol forming plants. In addition to sucrose and starch, Sorbitol is the primary photosynthetic end product in the leaves of many tree fruit species in the Rosaceae family. To understand the biochemical regulation of photosynthetic carbon partitioning between sorbitol, sucrose and starch in sorbitol synthesizing species, we purified cytoFB-Pase to apparent homogeneity from apple leaves. The enzyme was a homotetramer with a subunit mass of 37 kDa. It was highly specific for fructose-1,6-bisphosphate with a Km of 3.1 μm and a Vmax of 48 units/mg protein. Either Mg2+ or Mn2+ was required for its activity with a Km of 0.59 mm and 62 μM, respectively. Li+, Ca2+, Zn2+, Cu2+ and Hg2+ inhibited whereas Mn2+ enhanced the Mg2+-activated enzyme activity. Fructose-6-phosphate was found to be a mixed type inhibitor with a Ki of 0.47 mm. Fructose 2,6-bisphosphate (F2,6BP) competitively inhibited the enzyme activity and changed the substrate saturation curve from hyperbolic to sigmoidal. Adenosine monophosphate (AMP) was a non-competitive inhibitor for the enzyme. F2,6BP interacted with AMP to inhibit the enzyme in a synergistic way. Dihydroxyacetone phosphate did not have inhibitory effect on apple leaf cytosolic FBPase activity. Sorbitol increased the susceptibility of the enzyme to the inhibition by F1,6BP. The presence of sorbitol in the reaction mixture led to a reduction in the enzyme activity.

scholarly journals Metabolic control of hepatic gluconeogenesis during exercise

1983 ◽  
Vol 212 (3) ◽  
pp. 633-639 ◽  
Author(s):  
G L Dohm ◽  
E A Newsholme
Keyword(s):  
Metabolic Control ◽  
Pyruvate Kinase ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Prolonged Exercise ◽  
Allosteric Effectors ◽  
Fructose 1,6 Bisphosphatase ◽  
Metabolite Concentrations ◽  
Fructose 1,6 Bisphosphate ◽  
Hexose Phosphates

Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.

scholarly journals Starch degradation in chloroplasts isolated from C3 or CAM (crassulacean acid metabolism)-induced Mesembryanthemum crystallinum L

1996 ◽  
Vol 318 (3) ◽  
pp. 945-953 ◽  
Author(s):  
H. Ekkehard NEUHAUS ◽  
Norbert SCHULTE
Keyword(s):  
Crassulacean Acid Metabolism ◽  
Metabolic Flux ◽  
Acid Metabolism ◽  
Starch Content ◽  
Pentose Phosphate ◽  
Mesembryanthemum Crystallinum ◽  
Starch Degradation ◽  
Dihydroxyacetone Phosphate ◽  
Phosphoglyceric Acid ◽  
Isolated Chloroplasts

C3 or crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants perform nocturnal starch degradation which is linear with time. To analyse the composition of metabolites released by isolated leaf chloroplasts during starch degradation we developed a protocol for the purification of starch-containing plastids. Isolated chloroplasts from C3 or CAM-induced M. crystallinum plants are also able to degrade starch. With respect to the endogenous starch content of isolated plastids the rate of starch degradation in these organelles is close to the observed rates of starch degradation in intact leaves. The combined presence of Pi, ATP, and oxaloacetate is identified to be the most positive effector combination to induce starch mobilization. The metabolic flux through the oxidative pentose-phosphate pathway in chloroplasts isolated from CAM-induced M. crystallinum is less than 3.5% compared with other metabolic routes of starch degradation. Here we report that starch-degrading chloroplasts isolated from CAM-induced M. crystallinum plants use exogenously supplied oxaloacetate for the synthesis of malate. The main products of starch degradation exported into the incubation medium by these chloroplasts are glucose 6-phosphate, 3-phosphoglyceric acid, dihydroxyacetone phosphate and glucose. The identification of glucose 6-phosphate as an important metabolite released during starch degradation is in contrast to the observations made on all other types of plastids analysed so far, including chloroplasts isolated from M. crystallinum in the C3 state. Therefore, we analysed the transport properties of isolated chloroplasts from M. crystallinum. Surprisingly, both types of chloroplasts, isolated from either C3 or CAM-induced plants, are able to transport glucose 6-phosphate in counter exchange with endogenous Pi, indicating the presence of a glucose 6-phosphate translocator as recently demonstrated to occur in other types of plastids. The composition of metabolites released and the stimulatory effect of oxaloacetate on the rate of starch degradation are discussed with respect to the acidification observed for CAM leaves during the night.

scholarly journals Phosphonomethyl analogues of hexose phosphates

1976 ◽  
Vol 155 (2) ◽  
pp. 433-441 ◽  
Author(s):  
D Webster ◽  
W. R Jondorf ◽  
H B. F. Dixon
Keyword(s):  
Pentose Phosphate ◽  
Phosphate Group ◽  
Phosphogluconate Dehydrogenase ◽  
Fructose 1,6 Bisphosphatase ◽  
Pentose Phosphate Pathways ◽  
Fructose 1,6 Bisphosphate ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Hexose Phosphates

The analogue of fructose 1,6-bisphosphate in which the phosphate group, -O-PO3H2, on C-6 is replaced by the phosphonomethyl group, -CH2-PO3H2, was made enzymically from the corresponding analogue of 3-phosphoglycerate. It was a substrate for aldolase, which was used to form it, but not for fructose 1,6-bisphosphatase. It was hydrolysed chemically to yield the corresponding analogue of fructose 6-phosphate [i.e. 6-deoxy-6-(phosphonomethyl)-D-fructose, or, more strictly, 6,7-dideoxy-7-phosphono-D-arabino-2-heptulose]. This proved to be a substrate for the sequential actions of glucose 6-phosphate isomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Thus seven out of the nine enzymes of the glycolytic and pentose phosphate pathways so far tested catalyse the reactions of the phosphonomethyl isosteres of their substrates.

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