Metabolism in slices from growing potato tubers responds differently to addition of sucrose and glucose

The short-term changes in metabolism that occurred after adding glucose or sucrose to freshly cut discs from growing potato (Solanum tuberosum L.) tubers were investigated. (i) When glucose was supplied, there was a marked increase in glycolytic metabolites, and respiration was stimulated. When sucrose was supplied, amounts of glycolytic metabolites including hexose phosphates and 3-phosphoglycerate (3PGA) were similar to or lower than in control discs incubated without sugars, and respiration did not rise initially above that in control discs. This different response to sucrose and glucose was found across the concentration range 5–200 mM. A larger proportion of the metabolised ¹⁴C was converted to starch when [¹⁴C] sucrose was supplied than when [¹⁴C] glucose was supplied. The different effect on metabolite levels, respiration and starch synthesis was largest after 20–30 min, and decreased in longer incubations. (ii) When 5 or 25 mM sucrose was added in the presence of [¹⁴C] glucose, it led to a decrease in hexose phosphates and 3PGA, and a small increase in the rate of starch synthesis compared to discs incubated with glucose in the absence of sucrose. These differences were seen in a 30-min pulse and a 2-h pulse. Whereas ADP-glucose levels after adding sucrose resembled those in control discs, glucose led to a decrease in ADP-glucose. This decrease did not occur when 5 or 25 mM sucrose was added with the glucose. (iii) To check the relevance of these experiments for intact tubers, water or 100 mM mannitol, sucrose or glucose were supplied through the stolon to intact tubers for 24 h. A 0.2 mM solution of [¹⁴C] glucose was then introduced into the tubers, and its metabolism investigated during the next 30 min. Labelling of starch was increased after preincubation with sucrose, and significantly inhibited after preincubation with glucose. (iv) It is concluded that glucose and sucrose have different effects on tuber metabolism. Whereas glucose leads to a preferential stimulation of respiration, sucrose preferentially stimulates starch synthesis via a novel mechanism that allows stimulation of ADP-glucose pyrophosphorylase even though the levels of hexose phosphates and the allosteric activator 3PGA decrease. Received: 9 October 1997 / Accepted: 3 February 1998     

Orotate leads to a specific increase in uridine nucleotide levels and a stimulation of sucrose degradation and starch synthesis in discs from growing potato tubers

Freshly cut discs from growing potato tubers were incubated for 3 h with 10 mM orotate or 10 mM uridine. Control discs incubated without precursors showed a 30–40% decrease of uridine nucleotides, but not of adenine nucleotides. Orotate- and uridine-feeding led to a 1.5- to 2-fold increase in the levels of uridine nucleotides compared with control discs, and a 15–30% increase compared with the original values in intact tubers, but did not alter the levels of adenine nucleotides. Between 70–80% of the uridine nucleotides were present as UDPglucose, 15–25% as UTP, and 2–3% as UDP. The increase of uridine nucleotides involved a similar relative increase of UDPglucose, UTP and UDP. It was accompanied by a slight stimulation of the rate of [¹⁴C]sucrose uptake, a 2-fold stimulation of the rate at which the [¹⁴C]sucrose was subsequently metabolised, a small increase in the levels of hexose phosphates, glycerate-3-phospate and ADPglucose, and a 30% shift in the allocation of the metabolised label in favour of starch synthesis, resulting in a 2.4-fold stimulation of the rate of starch synthesis. Orotate led to a similar increase of uridine nucleotide levels in the presence of [¹⁴C]glucose, but did not significantly alter the rate of glucose uptake and metabolism to starch, nor did it increase the rate of sucrose resynthesis. The levels of uridine nucleotides were high in tubers on 6 to 10-week-old potato plants, and declined in tubers on 12 to 15-week-old plants. Comparison with the effect of the uridine nucleotide level in discs shows that the high levels of uridine nucleotides in tubers on young plants will play an important role in determining the rate at which sucrose can be converted to starch, and that the level of uridine nucleotides is probably co-limiting for sucrose-starch conversions in tubers on older plants. Received: 25 September 1998 / Accepted: 29 December 1998     

Regulation of sucrose and starch metabolism in potato tubers in response to short-term water deficit

To investigate the effect of water stress on carbon metabolism in growing potato tubers (Solanum tuberosum L.), freshly cut and washed discs were incubated in a range of mannitol concentrations corresponding to external water potential between 0 and −1.2 MPa. (i) Incorporation of [¹⁴C]glucose into starch was inhibited in water-stressed discs, and labeling of sucrose was increased. High glucose overrode the changes at low water stress (up to −0.5 MPa) but not at high water stress. (ii) Although [¹⁴C]sucrose uptake increased in water-stressed discs, less of the absorbed [¹⁴C]sucrose was metabolised. (iii) Analysis of the sucrose content of the discs confirmed that increasing water deficit leads to a switch, from net sucrose degradation to net sucrose synthesis. (iv) In parallel incubations containing identical concentrations of sugars but differing in which sugar was labeled, degradation of [¹⁴C]sucrose and labeling of sucrose from [¹⁴C]glucose and fructose was found at each mannitol concentration. This shows that there is a cycle of sucrose degradation and resynthesis in these tuber discs. Increasing the extent of water stress changed the relation between sucrose breakdown and sucrose synthesis, in favour of synthesis. (v) Analysis of metabolites showed a biphasic response to increasing water deficit. Moderate water stress (0–200 mM mannitol) led to a decrease of the phosphorylated intermediates, especially 3-phosphoglycerate (3PGA). The decrease of metabolites at moderate water stress was not seen when high concentrations of glucose were supplied to the discs. More extreme water stress (300–500 mM mannitol) was accompanied by an accumulation of metabolites at low and high glucose. (vi) Moderate water stress led to an activation of sucrose phosphate synthase (SPS) in discs, and in intact tubers. The stimulation involved a change in the kinetic properties of SPS, and was blocked␣by protein phosphatase inhibitors. (vii) The amount of ADP-glucose (ADPGlc) decreased when discs were incubated on 100 or 200 mM mannitol. There was a strong correlation between the in vivo levels of ADPGlc and 3PGA when discs were subjected to moderate water stress, and when the sugar supply was varied. (viii) The level of ADPGlc increased and starch synthesis was further inhibited when discs were incubated in 300–500 mM mannitol. (ix) It is proposed that moderate water stress leads to an activation of SPS and stimulates sucrose synthesis. The resulting decline of 3PGA leads to a partial inhibition of ADP-glucose pyrophosphorylase and starch synthesis. More-extreme water stress leads to a further alteration of partitioning, because it inhibits the activities of one or more of the enzymes involved in the terminal reactions of starch synthesis. Received: 26 August 1996 / Accepted: 5 November 1996     

Contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis is decreased by water stress in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch and cell-wall synthesis in tissue slices of growing potato (Solanum tuberosum L. cv. Desirée) tubers. Based on the analysis of fluxes and metabolites, Geigenberger et al. (1997, Planta 201: 502–518) proposed that water deficits up to −0.72 MPa stimulate sucrose synthesis, leading to decreased starch synthesis as a result of the resulting decline of phosphorylated metabolite levels, whereas more-severe water deficits directly inhibit the use of ADP-glucose. Potato plants with decreased expression of adenosine 5′-diphosphoglucose pyrophosphorylase (AGPase) have been used to test the prediction that the contribution of AGPase to the control of starch synthesis should decrease in severely water-stressed tuber material. Freshly cut slices from wild-type and antisense tubers were incubated at a range of mannitol concentrations (20, 300 and 500 mM) and the metabolism of [¹⁴C]glucose was analysed. A 86–97% reduction of AGPase activity led to a major but non-stoichiometric inhibition of starch accumulation in intact growing tubers attached to the plant (40–85%), and an inhibition of starch synthesis in non-stressed tuber slices incubated in 20 mM mannitol (60–80%). The inhibition of starch synthesis was accompanied by a 2- to 8-fold increase in the levels of sugars in intact tubers and a 2- to 3-fold stimulation of sucrose synthesis in tuber slices, whereas respiration and cell-wall synthesis were not significantly affected. The strong impact of AGPase on carbon partitioning in non-stressed tubers and tuber slices was retained in slices subjected to moderate water deficit (300 mM mannitol, corresponding to −0.72 MPa). In discs incubated in 500 mM mannitol (corresponding to −1.2 MPa) this response was modified. A 80–97% reduction of AGPase resulted in only a 0–40% inhibition of starch synthesis. Further, the water stress-induced stimulation of sucrose synthesis was abolished in the transformants. The results provide direct evidence that the contribution of AGPase to the control of starch synthesis can be modified by environmental factors, leading to a lower degree of control during severe water deficits. There was also a dramatic decrease in the labelling of cell-wall components in wild-type tuber slices incubated with 300 or 500 mM mannitol. The water stress-induced inhibition of cell-wall synthesis occurred independently of AGPase expression and the accompanying changes in starch and sucrose metabolism, indicating a direct inhibition of cell-wall synthesis in response to water stress. Received: 24 February 1999 / Accepted: 28 May 1999     

Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.     

Altered metabolic fluxes result from shifts in metabolite levels in sucrose phosphorylase-expressing potato tubers

As reported in a previous paper (Plant, Cell and Environment 24, 357–365, 2001), introduction of sucrose phosphorylase into the cytosol of potato results in increased respiration, an inhibition of starch accumulation and decreased tuber yield. Herein a more detailed investigation into the effect of sucrose phosphorylase expression on tuber metabolism, in order to understand why storage and growth are impaired is described. (1) Although the activity of the introduced sucrose phosphorylase was low and accounted for less than 10% of that of sucrose synthase its expression led to a decrease in the activities of enzymes of starch synthesis relative to enzymes of glycolysis and relative to total amylolytic activity. (2) Incubation of tuber discs in [¹⁴C]glucose revealed that the transformants display a two‐fold increase of the unidirectional rate of sucrose breakdown. However this was largely compensated by a large stimulation of sucrose re‐synthesis and therefore the net rate of sucrose breakdown was not greatly affected. Despite this fact major shifts in tuber metabolism, including depletion of sucrose to very low levels, higher rates of glycolysis, and larger pools of amino acids were observed in these lines. (3) Expression of sucrose phosphorylase led to a decrease of the cellular ATP/ADP ratio and energy charge in intact growing tubers. It was estimated that at least 30% of the ATP formed during respiration is consumed as a result of the large acceleration of the cycle of sucrose breakdown and re‐synthesis in the transformants. Although the absolute rate of starch synthesis in short‐term labelling experiments with discs rose, starch synthesis fell relative to other fluxes including respiration, and the overall starch content of the tubers was lower than in wild‐type tubers. (4) External supply of amino acids to replace sucrose as an osmoticum led to a feed‐back inhibition of glycolysis, but did not restore allocation to starch. (5) However, an external supply of the non‐metabolizable sucrose analogue palatinose – but not sucrose itself – stimulated flux to starch in the transformants. (6) The results indicate that the impaired performance of sucrose phosphorylase‐expressing tubers is attributable to decreased levels of sucrose and increased energy consumption during sucrose futile cycling, and imply that sucrose degradation via sucrose synthase is important to maintain a relatively large sucrose pool and to minimize the ATP consumption required for normal metabolic function in the wild type.     

Effects of adenine nucleotides on the Ca2+-gated cation channel in sarcoplasmic reticulum vesicles

The effects of nucleotides on the Ca2+-gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring choline influx. The choline influx was measured by following the change in scattered light intensity using the stopped flow technique. ATP enhanced the Ca2+-induced choline influx. The activation followed a single-site titration curve with a dissociation constant of 1.0 +/- 0.5 mM, independent of the Ca2+ concentration. ATP seems to increase the pore radius or number of channels without affecting the gating mechanism of the Ca2+-gated cation channel. ADP, AMP, and adenine enhanced the choline transport in a manner similar to ATP, but cAMP, ITP, UTP, CTP, and GTP did not. The apparent dissociation constants and the maximal activations were as follows: ATP 1.0 mM, 28-fold; ADP 0.9 mM, 18-fold; AMP 0.6 mM, 7-fold, and adenine 0.4 mM, 4-fold. Adenine and AMP behaved as a competitive inhibitor for the activation by ATP. These results are consistent with the Ca2+-induced Ca2+ release observed in skinned muscle fiber and isolated SR.     

Physiological level of norepinephrine increases adenine nucleotides hydrolysis in rat blood serum

Extracellular adenosine 5′-triphosphate (ATP) and its breakdown products, adenosine 5′-diphosphate (ADP) and adenosine, have significant effects on a variety of biological processes. NTPDase enzymes, responsible for adenine nucleotides hydrolysis, are considered the major regulators of purinergic signaling in the blood. Previous work by our group demonstrated that ATP and ADP hydrolysis in rat blood serum are higher during the dark (activity) phase compared to the light (rest) phase. In nocturnal animals (e.g., rats), important physiological changes occur during the dark phase, such as increased circulating levels of melatonin, corticosterone, and norepinephrine (NE). This study investigated the physiological effects, in vivo and in vitro, of melatonin, dexamethasone, and NE upon nucleotides hydrolysis in rat blood serum. For in vivo experiments, the animals received a single injection of saline (control), melatonin (0.05 mg/kg), dexamethasone (0.1 mg/kg), or NE (0.03 mg/kg). For in vitro experiments, melatonin (1.0 nM), dexamethasone (1.0 μM), or NE (1.0 nM) was added directly to the reaction medium with blood serum before starting the enzyme assay. The results demonstrated that ATP and ADP hydrolysis in both in vitro and in vivo experiments were significantly higher with NE treatment compared to control (in vitro: ATP = 36.63%, ADP = 22.43%, P < 0.05; in vivo: ATP = 44.1%, ADP = 37.28%, P < 0.001). No significant differences in adenine nucleotides hydrolysis were observed with melatonin and dexamethasone treatments. This study suggests a modulatory role of NE in the nucleotidases pathway, decreasing extracellular ATP and ADP, and suggests that NE might modulate its own release by increasing the activities of soluble nucleotidases.     

Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

Adenine nucleotides are of general importance for many aspectsof cell function, but their role in the regulation of biosyntheticprocesses is still unclear. It was previously reported thatdecreased expression of plastidial adenylate kinase, catalysingthe interconversion of ATP and AMP to ADP, leads to increasedadenylate pools and starch content in transgenic potato tubers.However, the underlying mechanisms were not elucidated. Here,it is shown that decreased expression of plastidial adenylatekinase in growing tubers leads to increased rates of respiratoryoxygen consumption and increased carbon fluxes into starch.Increased rates of starch synthesis were accompanied by post-translationalredox-activation of ADP-glucose pyrophosphorylase (AGPase),catalysing the key regulatory step of starch synthesis in theplastid, while there were no substantial changes in metabolicintermediates or sugar levels. A similar increase in post-translationalredox-activation of AGPase was found after supplying adenineto wild-type potato tuber discs to increase adenine nucleotidelevels. Results provide first evidence for a link between redox-activationof AGPase and adenine nucleotide levels in plants. Key words: Adenylate kinase, ADPglucose pyrophosphorylase, plastid, redox-regulation, potato, respiration, starch Received 18 September 2007; Revised 12 November 2007 Accepted 13 November 2007     

Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers

Overexpression of inorganic pyrophosphatase (PPase) from Escherichia coli in the cytosol of plants (ppa1 plants) leads to a decrease of inorganic pyrophosphate (PPi; U. Sonnewald, 1992, Plant J 2: 571–581). The consequences for sucrose-starch interconversions have now been studied in growing potato (Solanum tuberosum L. cv. Desirée) tubers. Sucrose is degraded via sucrose synthase and UDP-glucose pyrophosphorylase in growing tubers, and it was expected that the low PPi in the ppa1 transformants would restrict the mobilisation of sucrose and conversion to starch. Over-expression of PPase resulted in an accumulation of sucrose and UDP-glucose, and decreased concentrations of hexose phosphates and glycerate-3-phosphate in growing ppa1 tubers. Unexpectedly, the rate of degradation of [¹⁴C] sucrose was increased by up to 30%, the rate of starch synthesis was increased, and the starch content was increased by 20–30% in ppa1 tubers compared to wild-type tubers. Reasons for this unexpectedly efficient conversion of sucrose to starch in the ppa1 tubers were investigated. (i) The transformed tubers contained increased activities of several enzymes required for sucrose-starch interconversions including two- to threefold more sucrose synthase and 60% more ADP-glucose pyrophosphorylase. They also contained 30–100% increased activities of several glycolytic enzymes and amylase, increased protein, and unaltered or slightly decreased starch phosphorylase, acid invertase and mannosidase. (ii) The transformants contained higher pools of uridine nucleotides. As a result, although the UDP-glucose pool is increased two- to threefold, this does not lead to a decrease of UTP or UDP. (iii) The transformants contained twofold larger pools of ATP and ADP, and ADP-glucose was increased by up to threefold. In stored ppa1 tubers, there were no changes in the activities of glycolytic enzymes, and nucleotides did not increase. It is concluded that in growing tubers PPi has a wider significance than just being an energy donor for specific reactions in the cytosol. Increased rates of PPi hydrolysis also affect general aspects of cell activity including the levels of nucleotides and protein. Possible ways in which PPi hydrolysis could affect these processes are discussed. Received: 9 July 1997 / Accepted: 3 November 1997     

Subcellular distribution of adenine nucleotides in two Ehrlich cell lines metabolizing glucose

Adenine nucleotide compartmentation and cytochrome redox state were studied in two Ehrlich ascites tumor cell lines incubated in the presence of 5mM glucose to show differences between their energy metabolisms. Changes in the subcellular distribution of adenine nucleotides were very different in both cell lines. Glucose seemed to energize the less malignant cell line, as shown by the asymmetry of the ATP/ADP ratios between cytosolic and mitochondrial compartments, but this was not the case for the more malignant cell line. The cytochrome contents were similar in both cell lines, but the degree of cytochrome oxidation was higher in the less malignant cell line; this result is in agreement with a higher oxygen consumption in the less malignant cell line.     

Studies on adenine nucleotide exchange in mitochondria from the muscle tissue of Ascaris lumbricoides (Nematoda)

Adenine nucleotide exchange between the intra- and extramitochondrial compartments of mitochondria isolated from the muscle tissue of Ascaris lumbricoides was investigated. The exchange was specific for ATP and ADP, AMP, adenosine and non-adenine nucleotides were not exchanged at significant rates. All combinations of counter exchange were found to be possible between intra- and extramitochondrial ATP and ADP. Adenine nucleotide exchange in Ascaris muscle mitochondria was inhibited by atractyloside; was strongly temperature dependent; activated by potassium and magnesium and only slightly activated by calcium. The K_m for adenine nucleotide exchange in Ascaris mitochondria was 4·1 and 2·85 μm for ATP and ADP respectively. The properties of adenine nucleotide exchange in Ascaris muscle mitochondria are thus similar in general features to the adenine nucleotide translocase system of mammalian mitochondria.     

Carbohydrate metabolism during postharvest ripening in kiwifruit

Mature fruit (kiwifruit) of Actinidia deliciosa var. deliciosa (A. Chev.), (C.F.) Liang and Ferguson cv. Haywood (Chinese gooseberry) were harvested and allowed to ripen in the dark at 20° C. Changes were recorded in metabolites, starch and sugars, adenine nucleotides, respiration, and sucrose and glycolytic enzymes during the initiation of starch degradation, net starch-to-sucrose conversion and the respiratory climacteric. The conversion of starch to sucrose was not accompanied by a consistent increase in hexose-phosphates, and UDP-glucose declined. The activity of sucrose phosphate synthase (SPS) measured with saturating substrate rose soon after harvesting and long before net sucrose synthesis commenced. The onset of sugar accumulation correlated with an increase in SPS activity measured with limiting substrates. Throughout ripening, until sucrose accumulation ceased, feeding [¹⁴C] glucose led to labelling of sucrose and fructose, providing evidence for a cycle of sucrose synthesis and degradation. It is suggested that activation of SPS, amplified by futile cycles, may regulate the conversion of starch to sugars. The respiratory climacteric was delayed, compared with net starchsugar interconversion, and was accompanied by a general decline of pyruvate and all the glycolytic intermediates except fructose-1,6-bisphosphate. The ATP/ ADP ratio was maintained or even increased. It is argued that the respiratory climacteric cannot be simply a consequence of increased availability of respiratory substrate during starch-sugar conversion, nor can it result from an increased demand for ATP during this process.Abbreviations Frul,6bisP fructose-1,6-bisphosphate - Frul,6Pase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - SPS sucrose phosphate synthase - UDPGlc uridine 5'-diphosphoglucose We thank Professor G. Costa, University of Udine and Flavia Succhi, University of Bologna for their help in obtaining the fruit in Italy. E.A.M. was the recipient of a travel grant through the NZ/German Technological Agreement.     

Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

Purification and characterisation of a novel starch synthase selective for uridine 5′-diphosphate glucose from the red alga Gracilaria tenuistipitata

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-¹⁴C]glucose than with ADP[U-¹⁴C]glucose. The activity with UDP[U-¹⁴C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-¹⁴C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native M_r of 580 kDa and displays kinetic properties similar to other α-glucan synthases such as stimulation by citrate, product (UDP) inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed. Received: 29 January 1999 / Accepted: 11 March 1999     

The effect of exogenous sugars on the control of flux by adenosine 5′-diphosphoglucose pyrophosphorylase in potato tuber discs

The aim of this work was to investigate the effect of exogenous sugars on the extent to which starch synthesis in potato ( Solanum tuberosum L.) is controlled by adenosine 5'-diphosphoglucose pyrophosphorylase (EC 2.7.7.27; AGPase). Tuber discs were incubated in the presence of a range of concentrations of glucose and sucrose, and metabolic fluxes measured following the supply of [U-14C]glucose and measurement of the specific radioactivity of the hexose phosphate pool. In the presence of glucose there was a marked increase in the flux through glucose-phosphorylating hexokinase, and at high concentrations of external glucose this led to a stimulation of the rate of starch and sucrose synthesis relative to those measured in the presence of sucrose. In the presence of glucose the ratio of the rate of starch synthesis to the rate of glycolysis was higher than in the presence of sucrose. Similar effects of glucose were observed at two stages of tuber development. We conclude that the presence of glucose perturbs the carbohydrate metabolism of tuber discs so that starch synthesis is favoured. In order to determine the extent to which AGPase controls flux, we measured fluxes in wild-type plants and transgenic plants with reduced AGPase activity as a result of the expression of a cDNA encoding the B subunit in the antisense orientation. In the presence of sucrose a reduction in AGPase activity had a greater impact on the rate of starch synthesis than in the presence of glucose. The flux control coefficient of AGPase over starch synthesis was higher in the presence of sucrose (0.7-0.9) than in the presence of glucose (0.4-0.6). Conversely, the impact of reduced AGPase activity on the rate of sucrose synthesis was lower in the presence of sucrose than glucose. In the presence of 200 mM sucrose the flux control coefficient of AGPase over the rate of sucrose synthesis was not significantly different from zero. This demonstrates that the nature of the sugar supplied to potato tuber discs can have a major influence on the distribution of control within metabolism. These data were also used to investigate the relationship between demand for ATP and the rate of hexose phosphate entry into glycolysis. A very strong correlation between ATP demand and glycolytic flux was demonstrated.