Purification and properties of nitrite reductase from roots of pea (Pisum sativum cv. Meteor)

Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.Abbreviations DEAE diethylaminoethyl - EPR electron paramagnetic resonance - NIR nitrite reductase - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Genetic manipulation of 6-phosphofructokinase in potato tubers

The aim of this work was to discover whether genetic manipulation of 6-phosphofructokinase [EC 2.7.1.11; PFK(ATP)] influenced the rate of respiration of tuber tissue of Solanum tuberosum L. Transgenic plants were produced that contained the coding sequence of the Escherichia coli pfkA gene linked to a patatin promoter. Expression of this chimaeric gene in tubers resulted in a 14to 21-fold increase in the maximum catalytic activity of PFK(ATP) without affecting the activities of the other glycolytic enzymes. Tubers, and aged disks of tuber tissue, from transformed plants showed no more than a 30% fall in the content of hexose 6-monophosphates; the other intermediates of glycolysis increased threeto eightfold. Fructose-2,6-bisphosphate was barely detectable in aged disks of transformed tubers. The relative rates of ¹⁴CO₂ production from [1-¹⁴C]-and [6-¹⁴C]-glucose supplied to disks of transformed and control tubers were similar. Oxygen uptake and CO₂ production by aged disks of transformed tubers did not differ significantly from those from control tubers. The same was true of CO₂ production, in air, and in nitrogen, for tuber tissue. It is concluded that PFK(ATP) does not dominate the control of respiration in potato tubers.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - FW freshweight - GUS -glucuronidase - PFK(ATP) 6-phosphofructokinase - PFK(PPi) pyrophosphate: fructose-6-phosphate 1-phosphotransferase     

Ascorbate synthesis and ascorbate peroxidase activity during the early stage of wheat germination

Embryos from dry caryopses of wheat ( Triticum durum L. cv. Norba) are completely devoid of ascorbate (ASC) but contain a low amount of dehydroascorbate (DHA). The de novo biosynthesis of ASC starts in the wheat embryos after 8–10 h of germination but before the ASC biosynthetic pathway is completely restored the embryos can provide themselves with ASC by the reduction of the stored DHA. Three different proteins having DHA-reducing capability are present in the embryos during the early stages of germination. However, when the de novo ASC biosynthesis from sugar is completely restored, the DHA reduction capability largely drops and only one DHA-reducing protein remains active. The presence of three proteins having DHA-reducing capability and their behaviour during germination is discussed.
Dry embryos are also devoid of ASC peroxidase (EC 1.11.1.11); this hydrogen peroxide scavenger enzyme appears after the same lag as ASC and increases during germination in parallel with the rise in ASC. When ASC biosynthesis is experimentally induced, the ASC peroxidase also appears earlier; moreover the affinities for ASC of the three ASC peroxidase isoenzymes that progressively appear during germination depend on the ASC available in the embryos: highest in the first isoenzyme, that appears when the ASC content is very low, lowest in the isoenzyme that is expressed last, when the ASC content is 10–11 times higher.     

Effect of low temperature on the activity of phosphofructokinase from potato tubers

The aim of this work was to compare the coldlability of phosphofructokinase (EC 2.7.1.11) from tubers of potato cultivars (cvs.) known to differ in their propensity to accumulate sugars at low temperature. When stored at 4°C for six weeks, the sugar content of tubers ofSolanum tuberosum L. cv. Record doubled whereas the amount of sugar in tubers of cv. Brodick and an advanced breeding clone (13676) decreased slightly. Tubers from each line contained four forms of phophofructokinase. Over the range 12°–16°C the temperature coefficients of the four forms of phosphofructokinase from cvs. Record and Brodick were similar. In cv. Record the temperature coefficients of three of the enzyme forms were significantly higher at 2°–6°C than at 12°–16°C, whereas those from cv. Brodick were unchanged. These results are consistent with the proposal that inactivation of phosphofructokinase at low temperature results in the accumulation of hexose phosphates leading to increased sucrose synthesis.     

Protocol for DNA extraction from potato tubers

A method to extract high-quality DNA from potato tubers was developed and tested on 3 wild potato species (Solanum raphanifolium, S. megistracrolobum, S. bukasovii) and on the tetraploid B3 bred population, (population number 393228, derived fromS. tuberosum subsp.tuberosum). The average yield of extracted DNA varied from 10–30 μg of DNA per gram of processed tissue. The DNA was pure and suitable for ligation-mediated polymerase chain reaction (LM-PCR) amplification, producing clear, distinctive, and reproducible banding patterns in polyacrylamide gels.     

Sucrose uptake and partitioning in discs derived from source versus sink potato tubers

The uptake of sucrose into isolated discs cut from sink (growing) and source (sprouting) potato (Solanum tuberosum L.) tuber tissue was studied. The uptake of sucrose into sink-tuber discs demonstrated biphasic kinetics. The large saturable component was inhibited by incubation of the discs with p-chloromercuribenzene sulfonic acid (PCMBS) whilst both the saturable and linear components were inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP). By contrast, in source-tuber discs, the linear component represented the majority of sucrose taken up, the saturable component playing only a minor role. In source discs, only the saturable component of uptake was inhibited by either PCMBS or CCCP. A large proportion (up to 25%) of sucrose taken up into sink-tuber discs was converted to starch but as the tubers aged the proportion of sucrose converted to starch decreased to the level found in source-tuber discs (approx. 3%). By contrast with sink-tuber discs (see Oparka and Wright, 1988b, Planta 175, 520–526) sucrose uptake into source discs was insensitive to turgor and demonstrated an uptake pattern similar to that of CCCP-treated sink tissue. It is proposed that exogenous sucrose is taken into the storage parenchyma of sink-tuber discs by both a carrier-mediated and a diffusional process. By contrast, uptake into the storage parenchyma of source-tuber discs appears to be essentially diffusional. The turgor sensitivity of sucrose uptake into sink-tissue discs may be mediated via the plasmalemma H⁺-ATPase. As the tuber ages the sucrose-uptake activity decreases and the capacity of the storage parenchyma to synthesise starch is lost. The data are discussed in relation to the in-vivo mechanisms of sucrose transport in storage tissues.     

Effect of cadmium on glutathione reductase in potato tubers

Short-term treatment of potato tuber (Solanum tuberosum L.) discs with CdCl₂ changed glutathione reductase (GR) activity depending on cadmium ions concentrations, kind of tuber and time of incubation. The increase of GR activity at 10 and 100 μmol·dcm⁻³ of CdCl₂ solutions was marked in less resistant tissues of cv. Bintje after 24 hrs, and was slight in more resistant tissues of cv. Bzura after 72 hrs. At 1 mmol·dcm⁻³ concentration of CdCl₂ rapid and total inactivation in both kind of tissues was observed, which disappeared after a few days. However this elevation was faster in more resistant tissues. These inhibition effects come from the inactivation process of GR by cadmium. The values of K_I for cadmium and K_M for GSSG of GR from potato tuber tissues indicated that enzyme from more resistant tissues possessed lower affinity to toxic metal and higher affinity to substrate.     

Development of cyanide-resistant respiration in mitochondria from potato tubers treated with ethanol,acetaldehyde, and acetic acid

Treatment of intact potato (Solanum tuberosum L.) tubers with acetaldehyde, ethanol or acetic-acid vapors led to a respiratory upsurge which was further increased when the volatiles were applied in 100% O₂. Mitochondria from tubers held in 100% O₂ (O₂ control) displayed a substrate state, state 3, and state 4 in respiration, whereas in mitochondria from the volatile-treated tubers the respiratory rate of the different states was virtually indistinguishable. This respiratory pattern was companied by the development of a cyanide-resistant respiration since these mitochondria exhibited resistance to CN and sensitivity to CN+salicylhydroxamic acid. Acetaldehyde-treated potatoes showed a time-course development (up to 36 h) of cyanide resistance and concomitant sensitivity to salicylhydroxamic acid, indicating the onset of synthetic processes leading to the observed changes in mitochondrial respiration.Abbreviations V total respiration rate - V_cyt velocity of O₂ uptake attributable to cytochrome oxidase - V_alt velocity of O₂ uptake attributable to the alternate oxidase - RCR respiratory control ratio - SHAM salicylhydroxamic acid Paper of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, N.J., USA     

Synthesis and properties of glutathione reductase in stressed peas

We have subjected peas (Pisum sativum L.) to four different oxidative stresses: cold conditions (4 °C) in conjunction with light, treatment with paraquat, fumigation with ozone, and illumination of etiolated seedlings (greening). In crude extracts of leaves from stressed plants, an increase (up to twofold) in activity of glutathione reductase (GR) was observed which was consistent with previous reports from several laboratories. In all cases, except for ozone fumigation, the increase in activity was not due to an elevation in the steady-state levels of GR protein. None of the applied stresses had any effect on steady-state levels of GR mRNA. In contrast to the small increase in GR activity, the K _m of GR for glutathione disulphide showed a marked decrease when determined for extracts of stressed leaves, compared with that from unstressed plants. This indicates that GR from stressed plants has an increased affinity for glutathione disulphide. The profile of GR activity bands fractionated on non-denaturing acrylamide gels varied for extracts from differently stressed leaves and when compared with GR from unstressed plants. The changes in GR-band profiles and the alteration in the kinetic properties are best explained as changes in the isoform population of pea GR in response to stress.Abbreviations GR glutathione reductase - GSSG glutathione disulphide - Rubisco Ribulose-1,5-bisphosphate carboxylase-oxygenase - RNase A/T1 ribonucleases A and T1 We are grateful to Prof. Alan Wellburn and Dr. Phil Beckett (Division of Biological Sciences, University of Lancaster, UK) for providing ozone-fumigated material and Dr. Jeremy Harbinson for providing material grown at 4° C. This work was supported by a grant-in-aid to the John Innes Institute from the Agricultural and Food Research Council. E.A.E. and C.E. gratefully acknowledge the support of a John Innes Foundation studentship and a European Molecular Biology Organisation Fellowship respectively.     

The control of bud dormancy in potato tubers. Measurement of the seasonal pattern of changing concentrations of zeatin-cytokinins

A radioimmunoassay, combined with high-performance liquid chromatography, has been used to analyse the zeatin-type cytokinins of potato (Solanum tuberosum L. cv. Majestic) tubers and tuber buds throughout growth and storage. During tuber growth, zeatin riboside was the predominant cytokinin detected in all tissues. Immediately after harvest, the total cytokinin concentration fell dramatically in the storage tissue, largely as a consequence of the disappearance of zeatin riboside. During storage, levels of cytokinins in the storage tissue remained relatively constant, but increased in the tuber buds. In the buds of tubers stored at 2°C there was a 20-to 50-fold increase in total cytokinin over six weeks, coinciding with the natural break of innate dormancy. At 10°C the rise in the level of bud cytokinins was slower, correlating with the longer duration of innate dormancy. Injecting unlabelled cytokinins into tubers in amounts known to induce sprouting gave rise to increases in cytokinin concentrations in the buds of the same order as the increase associated with the natural break of dormancy. Metabolism of injected cytokinins was greater in non-dormant than in dormant tubers. The roles of cytokinin concentration and the sensitivity of the buds to cytokinin in the control of dormancy are discussed.Abbreviations CK cytokinin - FW fresh weight - HPLC high-performance liquid chromatography - RIA radioimmunoassay - tio⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purine=zeatin - tio⁶adeglc⁹ 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-glucopyranosyl purine=zeatin-9-glucoside - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosyl purine=zeatin riboside - tio⁶ado-[³H]-diol a radioactive derivative of zeatin riboside, synthesised by periodate-oxidation followed by [³H]NaBH₄-reduction - tio⁶AMP 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-5-phosphoribofuranosyl purine=zeatin riboside 5-monophosphate - t(ioglc⁴)⁶ade 6-(4-O--D-glucopyranosyl-3-methylbut-trans-2-enylamino)-purine=zeatin-O-glucoside     

Sucrose synthase catalyses a readily reversible reaction in vivo in developing potato tubers and other plant tissues

Experiments were carried out to investigate whether sucrose synthase (Susy) catalyses a readily reversible reaction in vivo in potato (Solanum tuberosum L.) tubers, Ricinus communis L. cotyledons, and heterotrophic Chenopodium rubrum L. cell-suspension cultures. (i) The contents of sucrose, fructose, UDP and UDP-glucose were measured and the mass-action ratio compared with the theoretical equilibrium constant. In all three tissues the values were similar. (ii) Evidence for rapid turnover of label in the sucrose pool was obtained in pulse-chase experiments with potato discs and with intact tubers attached to the plant. The unidirectional rates of sucrose synthesis and degradation were considerably higher than the net flux through the sucrose pool in the tubers. (iii) Labelling of the glucosyl and fructosyl moieties of sucrose from [¹⁴C]glucose in the presence of unlabelled fructose provided evidence that Susy contributes to the movement of label into sucrose. Methods for estimating the contribution of sucrose-phosphate synthase and Susy are presented and it is shown that their relative contribution varies. For example, the contribution of Susy is high in developing tubers and is negligible in harvested tubers which contain low Susy activity. (iv) The absolute values of the forward (v⁺¹) and backward (v^?1) reaction direction of Susy are calculated from the kinetic labelling data. The estimated values of v⁺¹ and v^?1 are comparable, and much higher than the net flux through the sucrose pool. (v) The estimated concentrations of the substrates and products of Susy in tubers are comparable to the published K _m values for potato-tuber Susy. (vi) It is concluded that Susy catalyses a readily reversible reaction in vivo and the relevance of this conclusion is discussed with respect to the regulation of sucrose breakdown and the role of Susy in phloem unloading.     

The control of bud dormancy in potato tubers

Potato (Solanum tuberosum L.) tuber buds normally remain dormant through the growing season until several weeks after harvest. In the cultivar Majestic, this innate dormancy persisted for 9 to 12 weeks in storage at 10° C, but only 3 to 4 weeks when the tubers were stored at 2° C. At certain stages, supplying cytokinins to tubers with innately dormant buds induced sprout growth within 2 d. The growth rate was comparable to that of buds whose innate dormancy had been lost naturally. Cytokinin-treatment did not accelerate the rates of cell division and cell expansion in buds whose innate dormancy had already broken naturally. Gibberellic acid did not induce sprout growth in buds with innate dormancy. We conclude that cytokinins may well be the primary factor in the switch from innate dormancy to the non-dormant state in potato tuber buds, but probably do not control the subsequent sprout growth.Abbreviations tio ⁶ade 6-(4-hydroxy-3-methylbut-trans-2-enyl amino)purine, zeatin - tio⁶ado 6-(4-hydroxy-3-methylbut-trans-2-enyl amino)-9--D-ribofuranosyl purine, zeatin riboside     

Purification and properties of dehydroascorbate reductase from spinach leaves

Dehydroascorbate reductase was detected in the leaves of several plants and has been partially purified from spinach leaves. The enzyme has a MW of ca 25 000, a pH optimum of 7.5, a K_m for glutathione (GSH) of 4.43 ± 0.4 mM and a K_m for dehydroascorbate of 0.34 ± 0.05 mM. High concentrations of dehydroascorbate inhibit the enzyme. Cysteine cannot replace GSH as a donor. The purified dehydroascorbate reductase is extremely unstable and also inhibited by compounds which react with thiol groups. Dehydroascorbate does not protect the enzyme against such inhibition. GSH reduces dehydroascorbate non-enzymically at alkaline pH values.     

Molecular identification of the ten subunits of cytochrome-c reductase from potato mitochondria

Cytochrome-c reductase (EC 1.10.2.2.) from Solanum tuberosum L. comprises ten subunits with apparent molecular sizes of 55, 53, 51, 35, 33, 25, 14, 12, 11 and 10 kDa on 14% SDS-PAGE. The identity of the subunits was analysed by direct amino-acid sequencing via cyclic Edman degradation. A large-scale purification procedure for the enzyme complex based on affinity chromatography and gelfiltraton is described. All subunits were enzymatically fragmented and the generated peptides were separated by reverse-phase HPLC. Complete or partial sequence determination of 33 peptides comprising a total of nearly 500 amino acids showed, that cytochrome-c reductase from potato contains three respiratory proteins (cytochrome b, cytochrome c ₁ and the Rieske iron-sulfur protein), four small proteins with molecular sizes below 15 kDa (so-called Q-binding, hinge, cytochrome-c ₁-linked and core-linked proteins) and three proteins in the 50-kDa range which show similarity to members of the core/PEP/MPP protein family (core/processing enhancing protein/mitochondrial processing peptidase). In fact these subunits show highest sequence identity either to MPP or PEP, which is in line with earlier findings, that isolated cytochrome-c reductase from potato exhibits processing activity towards mitochondrial precursor proteins.Abbreviations MPP mitochondrial processing peptidase - PEP processing enhancing protein This research was supported by the Deutsche Forschungsgemeinschaft.     

Purification of a flavoprotein having NADPH-cytochrome c reductase and transhydrogenase activities from Nitrobacter winogradskyi and its molecular and enzymatic properties

A flavoenzyme which showed NADPH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase EC 1.6.2.4) and transhydrogenase (NADPH-NAD⁺ oxidoreductase, EC 1.6.1.1) activities was purified to an electrophoretically homogeneous state from Nitrobacter winogradskyi. The reductase was a flavoprotein which contained one FAD per molecule but no FMN. The oxidized form of the enzyme showed absorption maxima at 272, 375 and 459 nm with a shoulder at 490 nm, its molecular weight was estimated to be 36,000 by SDS polyacrylamide gel electrophoresis, and the enzyme seemed to exist as a dimer in aqueous solution. The enzyme catalyzed reduction of cytochrome c, DCIP and benzylviologen by NADPH, oxidation of NADPH with menadione and duroquinone, and showed transhydrogenase activity. NADH was less effective than NADPH as the electron donor in the reactions catalyzed by the enzyme. The NADPH-reduction catalyzed by the enzyme of N. winogradskyi cytochrome c-550 and horse cytochrome c was stimulated by spinach ferredoxin. The enzyme reduced NADP⁺ with reduced spinach ferredoxin and benzylviologen radical.Abbreviations DCIP dichlorophenolindophenol - Tris trishydroxy-methylaminomethane - Mops 3-(N-morpholino) propanesulfonic acid - SDS sodium dodecylsufate     

The involvement of hydrogen peroxide in abscisic acid-induced activities of ascorbate peroxidase and glutathione reductase in rice roots

We have monitored the changes in antioxidant enzyme activities and H₂O₂ concentrations in roots of rice (Oryza sativa L., cv. Taichung Native 1) seedlings treated with exogenous abscisic acid(ABA). Decrease in superoxide dismutase (SOD) and catalase (CAT) activities was observed in rice roots in the presence of ABA. However, ascorbate peroxide (APX) and glutathione reductase (GR) activities were increased after the ABA treatment. ABA treatment resulted in an increase in H₂O₂ concentrations in rice roots. Pre-treatment with dimethylthiourea, a chemical trap for H₂O₂, and diphenyleneiodonium chloride (DPI), a well known inhibitor of NADPH oxidase, inhibited ABA-induced accumulation of H₂O₂ and ABA-induced activities of APX and GR. ABA-induced accumulation of H₂O₂ was found to be prior to ABA-induced activities of APX and GR. Our results suggest that H₂O₂ is involved in ABA-induced APX and GR activities in rice roots.     

Transport of ascorbate into plasma membrane vesicles ofPhaseolus vulgaris L.

Summary Incubation of bean hook plasma membrane vesicles in the presence of L-[¹⁴C]ascorbate (ASC) resulted in a specific recovery of significant levels of the ligand with the vesicles. The strong decrease in radioactive ASC detected after hypotonic disruption of the vesicles or after an assay at 4 °C indicated that ASC was probably transported from the medium into the lumen of the membrane vesicles. The concentration kinetics of this presumptive transport process revealed a saturation curve which best fitted a biphasic model. Each phase in this model showed Michaelis-Menten type kinetics. The kinetic parameters for the different phases were calculated to be 14 and 79 M (K _m1 andK _m2) and 26 and 53 pmol/min · mg protein (V _max1 andV _max2). High concentrations of iso-ascorbate, dehydroascorbate (DHA) or non-labelled ASC significantly reduced the uptake of the radioactive vitamin. It was demonstrated that sugar or amino acid carriers are not involved in the ASC transport reaction. Generation of transmembrane cation gradients (H⁺, K⁺, Ca²⁺, Na⁺) or addition of sulfhydryl reagents (pCMBS or NEM) did not affect the ASC uptake in any way. It is suggested that ASC is taken up by a facilitated diffusion mechanism.Abbreviations ASC ascorbate - DHA dehydroascorbate - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid     

Hexose metabolism in discs excised from developing potato (Solanum tuberosum L.) tubers

Metabolism of radiolabelled hexoses by discs excised from developing potato (Solanum tuberosum L.) tubers was been investigated in the presence of acid invertase to prevent accumulation of labelled sucrose in the bathing medium (Viola, 1996, Planta 198: 179–185). When the discs were incubated with either [U-¹⁴C]glucose or [U-¹⁴C]fructose without unlabelled hexoses, the unidirectional rate of sucrose synthesis was insignificant compared with that of sucrose breakdown. The inclusion of unlabelled fructose in the medium induced a dramatic increase in the unidirectional rate of sucroses synthesis in the tuber discs. Indeed, the decline in the sucrose content observed when discs were incubated without exogenous sugars could be completely prevented by including 300 mM fructose in the bathing medium. On the other hand, the inclusion of unlabelled glucose in the medium did not significantly affect the relative incorporation of [U-¹⁴C]glucose to starch, sucrose or glycolytic products. Substantial differences in the intramolecular distribution of ¹³C enrichment in the hexosyl moieties of sucrose were observed when the discs were incubated with either [2-¹³C]fructose or [2-¹³C]glucose. The pattern of ¹³C enrichment distribution in sucrose suggested that incoming glucose was converted into sucrose via the sucrose-phosphate synthase pathway whilst fructose was incorporated directly into sucrose via sucrose synthase. Quantitative estimations of metabolic fluxes in vivo in the discs were also provided. The apparent maximal rate of glucose phosphorylation was close to the extractable maximum catalytic activity of glucokinase. On the other hand, the apparent maximal rate of fructose phosphorylation was much lower than the maximum catalytic activity of fructokinase, suggesting that the activity of the enzyme (unlike that of glucokinase) was regulated in vivo. Although in the discs incubated with or without fructose the rates of starch synthesis or glycolysis were similar, the relative partitioning of metabolic intermediates into sucrose was much higher in discs incubated with fructose (0.6% and 32.6%, respectively). It is hypothesised that the equilibrium of the reaction catalysed by sucrose synthase in vivo is affected in discs incubated with fructose as a result of the accumulation of the sugar in the tissue. This results in the onset of sucrose cycling. Incubation with glucose enhanced all metabolic fluxes. In particular, the net rate of starch synthesis increased from 2.0 mol · hexose · g FW^–1 · h^–1 in the absence of exogenous glucose to 3.7 mol · hexose · g FW^–1 · h^–1 in the presence of 300 mM glucose. These data are taken as an indication that the regulation of fructokinase in vivo may represent a limiting factor in the utilisation of sucrose for biosynthetic processes in developing potato tubers.Abbreviations ADPGlc adenosine 5-diphosphoglucose - Glc6P glucose-6-phosphate - hexose-P hexose phosphate - NMR nuclear magnetic resonance - UDPGlc uridine 5-diphosphoglucose Many thanks to L. Sommerville for skillfull assistance and to J. Crawford and J. Liu for useful discussions on flux analysis. The research was funded by the Scottish Office Agriculture and Fisheries Department.     

Sucrose-phosphate synthase is regulated via metabolites and protein phosphorylation in potato tubers,in a manner analogous to the enzyme in leaves

(i) Sucrose-phosphate synthase (SPS) was purified 40-fold from stored potato (Solanum tuberosum L.) tubers to a final specific activity of 33–70 nkat·(mg protein)^–1 via batch elution from diethylaminoethyl (DEAE)-sephacel, polyethylene glycol (PEG) precipitation and Mono Q anion-exchange chromatography. (ii) Immunoblotting revealed a major and a minor band with molecular weights of 124.8 kDa and 133.5 kDa, respectively. Both bands were also present in extracts prepared in boiling SDS to exclude proteolysis. No smaller polypeptides were seen, except when the preparations were incubated before application on a polyacrylamide gel. (iii) The enzyme preparation was activated by glucose-6-phosphate and inhibited by inorganic phosphate. Both effectors had a large effect on the K _m (fructose-6-phosphate) and the K _m (uridine-5-diphosphoglucose) with phosphate acting antagonistically to glucose-6-phosphate. (iv) Preincubation of potato slices with low concentrations of okadaic acid or microcystin resulted in a three- to fourfold decrease in the activity of SPS when the tissue was subsequently extracted and assayed. The decrease was especially marked when the assay contained low concentrations of substrates and glucose-6-phosphate, and inorganic phosphate was included. Preincubation with mannose or in high osmoticum resulted in an increase of SPS activity. (v) Analogous changes were observed in germinating Ricinus communis L. seedlings. After preincubation of the cotyledons in glucose, high SPS activity could be measured, whereas okadaic acid, omission of glucose, or addition of phosphate or sucrose led to a large decrease of SPS activity in the selective assay. (vi) It is argued that SPS from non-photosynthetic tissues is regulated by metabolites and by protein phosphorylation in an analogous manner to the leaf enzyme.Abbreviations Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - Pi inorganic phosphate - PGI phosphoglucose isomerase - PP2A phosphoprotein phosphatase 2A - PEG polyethyleneglycol - SPS sucrose-phosphate synthase - UDPGlc uridine-5-diphosphoglucose This work was supported by the Deutsche Forschungsgemeinschaft, the BMFT and Sandoz AG, Basel, Switzerland. We are grateful to Prof. E. Beck (Pflanzenphysiologie, Bayreuth, Germany) for providing us with laboratory facilities, and to Dr. U. Sonnewald (Institut für Genbiologische Forschung, Berlin, Germany) for many discussions and providing us with unpublished data.