Biochemical properties of potato phosphorylase change with its intracellular localization as revealed by immunological methods

Phosphorylase was purified from young and senescent potato tubers. Antibodies raised against the enzyme from young tubers crossreacted with phosphorylase from old tissue, although the latter exhibited different physico-chemical properties. In polyacrylamide gel electrophoresis it migrated with higher mobility, its subunit molecular weight was determined in the range of 40,000 in contrast to 100,000 of the phosphorylase in young tubers. The enzyme of senescent tubers displayed an isoelectric point of 5.4 different from the one of young tubers with 5.0, and the diffusion coefficients of the two enzymes varied. The appearance of the phosphorylase form typical for senescent tissue is connected with changes in the intracellular localization as revealed by immunofluorescence. Before massive starch accumulation is initiated, non-vacuolated subepidermal cells contain antigenically active material in their cytoplasm. During starch accumulation in fully differentiated storage parenchyma, only amyloplasts fluoresce, indicating the presence of adsorbed phosphorylase protein. Cytoplasmic phosphorylase can be detected in the continuance of senescence and, finally, after 16 months of tuber storage, the particle-bound enzyme had mostly disappeared. Simultaneously, we observed membrane destruction and decomposition on the ultrastructural level. The phosphorylase from senescent potatoes is a converted molecule and seems to be formed by proteolytic cleavage. The location of phosphorylase in the amyloplasts during starch synthesis indicates that it also plays a role in starch synthesis and not only in its degradation.Abbreviations PBS phosphate buffered saline - FITC fluorescein-isothiocyanate - IgG immunoglobuline G Dedicated to Professor Dr. A. Frey-Wyssling on the occasion of his 80th birthday     

Mechanism of the phosphorylase reaction. Utilization of D-gluco-hept-1-enitol in the absence of primer

alpha-Glucan phosphorylases from rabbit skeletal muscle, potato tubers and Escherichia coli catalyze the utilization of 2,6-anhydro-1-deoxy-D-gluco-hept-1-enitol (heptenitol) in the presence of arsenate or phosphate. 1H-NMR analysis in the presence of 2H2O and arsenate indicated formation of 1-[1-2H]deoxy-alpha-D-glucoheptulose with rates comparable to the arsenolysis of poly- or oligosaccharides. The reaction depends on the presence of a dianionic 5'-phosphate group of pyridoxal in the active conformation of the phosphorylases. Heptenitol is the first known substrate of alpha-glucan phosphorylases which does not require a primer. This is explained by the finding that heptenitol is exclusively used as substrate for the degradative pathway of the phosphorylase reaction where it competes with polysaccharide substrates. In the presence of phosphate the reaction product is 1-deoxy-alpha-D-gluco-heptulose 2-phosphate (heptulose-2-P), which subsequently inhibits the reaction. This characterizes heptulose-2-P as an enzyme-derived inhibitor. The Ki = 1.9 X 10(-6) M with potato phosphorylase suggests the formation of a transition-state-like enzyme-ligand complex. These findings, together with the fact that the phosphates of heptulose-2-P and pyridoxal 5'-phosphate are linked by hydrogen bridges [Klein, H. W., Im, M. J., Palm, D. & Helmreich, E. J. M. (1984) Biochemistry 23, 5853-5861], make it likely that both phosphates are involved in phosphorylase catalysis. A catalytic mechanism of phosphorylase action is proposed in which a 'mobile' phosphate anion plays a versatile role. It serves as proton carrier for the substrate activation, it stabilizes the intermediate and acts as a nucleophile which can accept a glycosyl residue reversibly.     

Inhibition Pattern of Beet-Saponin on Amylose Formation by Potato Phosphorylase

By using the multiple ascent technique, the authors have resolved the first several oligosaccharides of the product of initial stage of potato phosphorylase action in both the absence and the presence of beet-saponin with maltotriose as primer. The resolved chromatogram was sprayed with a mixture of G-1-P and phosphorylase, followed by spraying with iodine solution to locate the spots in which starch synthesis occurred.

Multi-chain mechanism of amylose formation from maltotriose and the suppression of lengthening of amylose chain by beet-saponin in the lag stage of enzyme action could be shown on paper chromatogram. No saccharides other than amylose series were recognized in the case of phosphorylase inhibition by beet-saponin.     

Translucent Tissue Defects in Solanum tuberosum L: I. Alterations in Amyloplast Membrane Integrity, Enzyme Activities, Sugars, and Starch Content

Kennebec (cv) potatoes randomly developed translucent areas in their centrally located pith-parenchymal cells during storage. These defective areas were characterized as having reduced starch concentration and increased levels of free sugars (i. e. sucrose and glucose) and inorganic phosphate. Electron micrographs of potato tubers stored at 10° ± 1°C for 8 months indicated that the amyloplast membrane was still intact and continuous around starch granules in both normal and prematurely sweetened tissue. The total activities of phosphorylase and sucrose-6-P synthase were elevated 5.4- and 3.8-fold, respectively, in the defective tissue compared to healthy nonsweetened tubers while there were no significant differences in the levels of sucrose synthase, UDPglucose pyrophosphorylase, invertase, or α-amylase. Total and specific activities of acid phosphatase were only slightly elevated in translucent tissue but their increase was significant (P < 0.05, t test) over that seen in healthy tubers. The premature sweetening in storage may have been indirectly triggered by moisture and heat stress experienced during development. Translucency eventually led to physical deterioration of the tissue.     

Activity of phosphorylase in Solanum tuberosum during low temperature storage

Changes in the activity of phosphorylase were measured during storage of potatoes at + 2° when the sugar content rises rapidly and subsequently at + 10° when the accumulated sugar is converted mainly to starch. The observed changes were relatively small and could not be related to any of the components of the phosphorylase system, which was shown to be complex.     

Formation of a soluble amylopectin-like polysaccharide in potato tubers

When potato sprouts or potato tuber slices were incubated with 0.1 m glucose 1-phosphate, a soluble amylopectin-like polysaccharide was excreted to the medium. This polysaccharide was found to be a very good primer for phosphorylase and a poor one for starch synthetase. Beside the formation of this extracellular polysaccharide, a more branched intracellular polysaccharide could be isolated. This polysaccharide was an excellent primer for starch synthetase. Fructose 6-phosphate, glucose 6-phosphate, fructose 1,6-diphosphate, glucose or sucrose could not substitute for glucose 1-phosphate. 2,4-Dinitrophenol or nitrogen did not affect the excretion of the polysaccharide. Some properties of these 2 polysaccharides are described.     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

Enzymes of starch metabolism in developing grains of high lysine barley mutant

The absolute activities of ADPG(UDPG)-pyrophosphorylase, starch phosphorylase, ADPG(UDPG)-starch synthetase, NDP-kinase and inorganic pyrophosphatase have been studied in high lysine mutant barley Notch-2 and its parent NP 113 grains during development. In general, mutant Notch-2 grains had higher average activities of UDPG-pyrophosphorylase and starch phosphorylase and lower activity of ADPG(UDPG)-starch synthetase per grain than the parent NP 113 during grain development. Activities of NDP-kinase, ADPG-pyrophosphorylase and inorganic pyrophosphatase differed only to a small extent between the mutant Notch-2 and NP 113. It is suggested that the lower activity of ADPG(UDPG)-starch synthetase might be responsible for the reduced accumulation of starch in the mutant Notch-2 grain as compared with parent NP 113 during development.     

The control of glycogen metabolism in yeast. 2. A kinetic study of the two forms of glycogen synthase and of glycogen phosphorylase and an investigation of their interconversion in a cell-free extract

Two interconvertible forms of glycogen synthase and glycogen phosphorylase, one active (a) or the other less active (b), were predominantly present in a thermosensitive adenylate-cyclase-deficient mutant that had been preincubated at the restrictive temperature of 35 degrees C, either in the presence or in the absence of glucose. Glycogen phosphorylase was at least 20-fold less active after incubation of the cells in the presence of glucose, but this residual activity had kinetic properties identical to those of the active form of enzyme, obtained after incubation in the absence of glucose; this suggests that the b form might be completely inactive and that the low activity measured after glucose treatment must be attributed to a residual amount of phosphorylase a. By contrast, the kinetic properties of the two forms of glycogen synthase were very different. When measured in the absence of glucose 6-phosphate, the two forms of enzyme had a similar affinity for UDP-Glc but differed essentially by their Vmax. Glucose 6-phosphate had no effect on synthase a, but increased both Vmax and Km of synthase b; these effects, however, were in great part counteracted by sulfate and by inorganic phosphate, the latter also having the property of increasing the Km of the a form, without affecting Vmax. It was estimated that at physiological concentrations of substrates and effectors, synthase a was about 20-fold more active than synthase b. When an extract of cells that had been preincubated in the absence of glucose was gel-filtered and then incubated at 30 degrees C, phosphorylase was progressively fully inactivated and synthase was partially activated; these reactions were severalfold faster and, in the case of glycogen synthase, more complete in the presence of 10 mM glucose 6-phosphate. When a gel-filtered extract of cells that had been preincubated in the presence of glucose was incubated at 30 degrees C in the presence of ATP-Mg and EGTA, phosphorylase became activated and synthase was inactivated; the first of these two reactions was severalfold stimulated by micromolar concentrations of Ca2+, whereas both reactions were completely inhibited by 10 mM glucose 6-phosphate and only slightly and irregularly stimulated by cyclic AMP.     

Mechanism of Purine Arabinoside Synthesis by Bacterial Transarabinosylation Reaction

The mechanism of purine arabinoside synthesis from uracil arabinoside and purine bases via the bacterial transarabinosylation reaction was investigated. Arabinose-1-phosphate was isolated from the reaction mixture in the form of the barium salt and proved to be the intermediate of the reaction. Two enzyme fractions were obtained from Enterobacter aerogenes by means of heat treatment, ammonium sulfate fractionation and DEAE-cellulose column chromatography. One enzyme split uracil arabinoside into uracil and arabinose-1-phosphate in the presence of inorganic phosphate and the other synthesized hypoxanthine arabinoside from arabinose-1-phosphate and hypoxanthine. The substrate specificity of these enzymes indicated that the former was uridine phosphorylase and the latter was purine nucleoside phosphorylase, respectively. Hypoxanthine arabinoside was synthesized from uracil arabinoside and hypoxanthine only in the presence of both enzymes and inorganic phosphate.     

Phosphate positioning and availability in the starch granule matrix as studied by EPR

Cu(2+) was introduced as an EPR probe into the starch granules isolated from different starch crop genotypes including transgenically modified potatoes generated for extreme amylose and starch phosphate monoester concentrations. Several discrete copper adducts bound to the starch matrix with different strength was revealed. It was found that phosphate has a significant influence on the type of these species, their number, location in the structure, and strength of binding. Well dispersed Cu(2+) complexes with axial symmetry are formed in the semicrystalline part of the starch linked through O-P- bonds in the phosphorylated starches. In the amorphous part of the starch, freely rotating hexaaqua complexes of Cu(2+) and complexes coupled antiferromagnetically are formed. The amount of the former increases with content of phosphate indicating enhanced binding of water in the granules. The results complement previous experimental data and molecular models for the starch granule with respect to the location and effects of phosphate and crystalline matter.     

Oxyanion-Mediated Protein Stabilization: Differential Roles of Phosphate for Preventing Inactivation of Bacterial α-Glucan Phosphorylases

Maltodextrin phosphorylase (MalP) from Escherichia coli and starch phosphorylase (StP) from Corynebacterium callunae are significantly stabilized in the presence of phosphate against inactivation by elevated temperature or urea. The stabilizing effect of phosphate was observed at ion concentrations below 50 mM. Therefore, it is probably due to preferential binding of phosphate to the folded conformations of the phosphorylases. For StP, phosphate binding inhibited the dissociation of the active-site cofactor pyridoxal 5′-phosphate. Phosphate-liganded StP was at least 500-fold more stable at 60d`C than the free enzyme at the same temperature. It showed an apparent transition midpoint of 5.2 M for irreversible denaturation by urea, and this midpoint was increased by a denaturant concentration of 4M relative to the corresponding transition midpoint of free StP in urea. The mechanisms of inactivation and denaturation of MalP at 45d`C and by urea involve formation of a cofactor-containing, insoluble protein aggregate. Under denaturing conditions, phosphate was shown to inhibit aggregation of the reversibly inactivated MalP dimer.     

Evidence for a new intermediate state in the mechanism of (Na+ + K+)-adenosine triphosphatase.

A rapid mixing technique was used to follow the intermediate formation of phosphorylated enzyme and liberation of inorganic phosphate by a microsomal preparation of (Na+ + K+)-ATPase. In the presence of 100 mM Na+,but without added K+, phosphorylation reaches a constant level at a rate which is dependent on ATP concentration. Inorganic phosphate production lags during the inital phase of phosphorylation and then accumulates at a constant rate. These observations favor a scheme in which Pi is liberated as the result of turnover of the phosphorylated enzyme. In the presence of 100 mM Na+ and 2.5 mM K+ phosphate production was resolved into two phases consisting of an initial 'burst' and late steady state phase...     

The citrate-stimulated starch synthase of starchy maize kernels: Purification and properties

Chromatography of maize kernel extracts on DEAE-cellulose resolves two fractions of starch synthase activity, one of which (starch synthase 1) is capable of synthesizing α-glucan in the absence of exogenous primer and the presence of 0.5 m citrate (J. L. Ozbun, J. S. Hawker, and J. Preiss, Plant Physiol. (1971) 48, 765–769). This starch synthase has been purified 200-fold from developing kernels of normal maize, and shown to have no detectable activities of branching enzyme, amylase, pullulanase, phosphorylase, and D enzyme. The preparation, however, was not electrophoretically homogeneous. This preparation had a K_m value of 0.033 mm for ADPglucose in the presence of 0.5 m citrate. The reaction in the presence of citrate was stimulated 10-fold by the addition of excess purified branching enzyme. This stimulation is higher than those reported previously (C. D. Boyer and J. Preiss, Plant Physiol. (1979) 64, 1039–1042) but is consistent with the predicted effects of removal of amylase activity. The effects of salts other than citrate on activity in the absence of exogenous primer were small, but the stimulation could be restored by the addition of excess purified branching enzyme. Citrate increased the affinity of the enzyme for the endogenous primer present to such a level that no effect of exogenous primer on reaction rate could be observed in the presence of 0.5 m citrate. Analysis of the glucan/iodine complex and the enzymatic breakdown products patterns from the products of the starch synthase reaction indicates a high degree of linearity. The results obtained are discussed in relation to the biosynthesis of starch in vivo.     

Enzymes of starch and sucrose metabolism in Zea mays leaves

Mesophyll and bundle sheath cells of maize leaves were separated and enzymes of starch and sucrose metabolism assayed. The starch content and activities of ADPglucose (ADPG) starch synthetase and phosphorylase expressed both on a chlorophyll and a protein basis were much lower in mesophyll cells compared to bundle sheath preparations. Exposure of the leaves to continuous illumination for 2·5 days caused the starch content of mesophyll cells to rise greatly and led to considerable increases in ADPG starch synthetase and phosphorylase activity. In glasshouse grown leaves the bulk of invertase, sucrose phosphate synthetase, sucrose phosphatase, UDPglucose pyrophosphorylase and amylase was situated in the mesophyll layer. Sucrose synthetase, ADPG starch synthetase and phosphorylase were largely confined to the bundle sheath. No enzyme could be completely assigned to one particular cell layer. Upon continuous illumination both ADPG starch synthetase and phosphorylase increased in the mesophyll bythe same relative amount. The mesophyll is likely to be a major site for sucrose synthesis in maize leaves.     

A novel sucrose phosphorylase from the metagenomes of sucrose-rich environment: isolation and characterization

Sucrose phosphorylase, an important enzyme mainly involved in the generic starch and sucrose pathways, has now caught the attention of researchers due to its transglycosylation activity. A novel sucrose phosphorylase, unspase, has been isolated, and its transglycosylation properties were characterized. Compared with Bisp, the sucrose phosphorylase from Bifidobacterium adolescentis, unspase had two deleted regions in its C: -terminal. These deleted regions were probably equivalent to the important five-stranded anti-parallel β-sheet domain in sucrose phosphorylase. Unspase has a k(m) of 21.12?mM, a V(max) of 69.24?μmol?min(-1)?mg(-1) and a k(cat) of 31.19?s(-1) with sucrose as substrate. In 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, unspase transferred the glycosyl moiety to L: -arabinose, D: -fructose and L: -sorbose. Much to our surprise, unspase can catalyze the transglycosylation in which a glycosyl moiety was transferred to L: -arabinose in the presence of phosphate, which is an interesting exception to the generally accepted fact that transglycosylation can only occur under the condition of phosphate absence. The final yield of the transglycosylation product (37.9?%) in phosphate buffer was even higher than that (5.8?%) in MOPS buffer. This is a novel phenomenon that a sucrose phosphorylase can catalyze a transglycosylation reaction in the presence of phosphate.     

Amylopectin degradation in pea chloroplast extracts

Phosphorolysis rather than phosphorylation of amylolysis products was found to be the major pathway of sugar phosphate formation from amylopectin by pea (Pisum sativum L.) chloroplast stromal proteins. The K_m for inorganic phosphate incorporation was 2.5 mm, and ATP did not stimulate amylopectin-dependent phosphate incorporation. Arsenate (10 mm) inhibited phosphate incorporation into glucose monophosphates up to 46% and phosphoglucomutase activity 96%, resulting in glucose 1-phosphate accumulation as a product of amylopectin degradation. The intracellular distribution of enzymes of starch utilization was determined. Phosphorylase, phosphoglucomutase, and hexokinase were found in the chloroplast and cytoplasm, while β-amylase was restricted to the cytoplasm. Maltase was not detectable; maltose phosphorylase was active in the chloroplast.     

Changes in the microtubular cytoskeleton precede in vitro tuber formation in potato

Summary An in vitro system for tuber formation was used to study early morphological and cytological changes occurring during tuber formation in potatoes, with special emphasis on the orientation of the microtubular cytoskeleton, visualized immunocytochemically. Axillary buds from potato plants were cultured in the presence or absence of gibberellin (GA), resulting in either tuber formation (without GA) or shoot formation (GA added). Tuber formation in the absence of GA was highly synchronous in individual buds, enabling the dissection of various aspects of tuberization. Under both conditions, starch started to accumulate. In the absence of GA, starch levels rapidly increased, concomitantly with tuber formation, whereas it slightly decreased in the presence of GA. Up to 4 days, the cortical MTs in the cells were oriented perpendicular to the longitudinal axis of the developing buds. Under tuber-inducing conditions this orientation changed into a longitudinal one at day 5. This change preceded a change in the direction of cell expansion. In the presence of GA no such reorientation was observed, cells continued to grow longitudinally, and a stoloniferous shoot was formed. The cytoskeletal changes preceded the visible swelling of the buds, observed after day 5, demonstrating that the reorientation of the microtubular cytoskeleton is one of the earliest steps observed so far in tuber formation in potatoes.Abbreviations GA gibberellin - MTs microtubules - PBS phosphate buffered saline - SD short-day     

The composition of the expressed sap from cold stored potatoes

Analysis of the chemical composition of expressed sap from potatoes stored at low temperatures (2°) indicates that an increased amount of inorganic phosphate is present in the juice from the cytoplasm and vacuole.     

Critical importance of phytase for yeast growth and alcohol fermentation in Japanese sake brewing

The high phytase producing mutant of Aspergillus oryzae (KL-38) previously isolated was employed for koji making, and the produced koji rice then supplied for sake brewing. The alcohol fermentation was improved compared to that with the parent strain (A. oryzae BP-1). The effects of two phytase isozymes (Phy I and Phy II) produced by A. oryzae on yeast growth and inorganic phosphate liberation were investigated using a synthetic medium containing phytic acid as a sole phosphate source. Yeast growth and the liberation of inorganic phosphate were both enhanced by the combination of Phy I and Phy II at a ratio of 1 to 3, which was compatible with the production ratio in KL-38. Based on these results, phytase plays important role in sake brewing, and that the maximum inorganic phosphate liberation from phytic acid can be obtained by a suitable combination of Phy I and Phy II.