Potato Tuber UDP-Glucose:Protein Transglucosylase Catalyzes Its Own Glucosylation

Potato (Solanum tuberosum L.) tuber UDP-glucose:protein transglucosylase (UPTG) (EC 2.4.1.112) is involved in the first of a two-step mechanism proposed for protein-bound α-glucan synthesis by catalyzing the covalent attachment of a single glucose residue to an acceptor protein. The resulting glucosylated 38-kilodalton polypeptide would then serve as a primer for enzymic glucan chain elongation during the second step. In the present report, we describe the fast protein liquid chromatography purification of UPTG from a membrane pellet of potato tuber. An apparently close association of UPTG, phosphorylase, and starch synthase was observed under native conditions during different purification steps. Enrichment of a 38-kilodalton polypeptide was found throughout enzyme purification. It is now shown that the purified UPTG, with an apparent molecular mass of 38 kilodaltons, undergoes self-glucosylation in a UDP-glucose- and Mn²⁺-dependent reaction. Therefore, it is concluded that UPTG is the enzyme and at the same time the priming protein required for the biogenesis of protein-bound α-glucan in potato tuber.     

Starch phosphorylase inhibitor from sweet potato

A protein, starch phosphorylase inhibitor, was purified from the root of sweet potato (Ipomoea batatas [L.] Lam. cv Tainon 65). It had a molecular weight of 250,000 and could be composed of five identical subunits. The isoelectric point of the inhibitor was 4.63. It was a noncompetitive inhibitor toward the sweet potato enzyme with a K_i value of 1.3 × 10⁻⁶ molar when glucose-1-P was the variable substrate. Because cross-reacting materials of rabbit antiphosphorylase inhibitor of sweet potato were found in three arbitrarily selected plant materials, viz. potato tuber, spinach leaf, and rice grain, the occurrence of this protein seemed universal in higher plants. By an immunofluorescence technique, the inhibitor was located in the amyloplast and cell wall where phosphorylase was also found. This implies that they may interact in vivo, and the inhibitor may play an unknown regulatory role against the plant enzyme.     

Purification and properties of hypoxically induced lactate dehydrogenase from barley roots

Using Affigel Blue and oxamate-agarose affinity chromatography, lactate dehydrogenase (LDH) was purified 2000-fold from hypoxically induced barley roots. Molecular weights of the native and sodium dodecyl sulfate-denatured LDH protein were 157 and 40 kilodaltons, respectively, indicating a tetramer. Purified barley LDH was very similar in size and kinetic properties to potato LDH. However, their amino acid compositions differed substantially and antibodies raised against barley LDH did not cross-react with potato LDH on immunoblots, implying that the barley and potato LDHs are not closely related proteins. In vivo [³⁵S] methionine labeling and immunoprecipitation experiments indicated that hypoxia increased the rate of LDH protein synthesis, and immunoblot analysis showed that LDH protein levels rose during hypoxia. We conclude that increased enzyme synthesis plays a major part in the induction of LDH enzyme activity by low O₂ levels in barley roots.     

Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.     

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.     

Uridine 5′-Diphosphate-Glucose Dehydrogenase from Soybean Nodules

A highly purified preparation of uridine 5′-diphosphate (UDP)-glucose (Glc) dehydrogenase (DH; EC 1.1.1.22) has been characterized from soybean (Glycine max L.) nodules. The enzyme had native and subunit molecular masses of approximately 272 and 50 kD, respectively. UDP-Glc DH displayed typical hyperbolic substrate kinetics and had K_m values for UDP-Glc and NAD⁺ of 0.05 and 0.12 mm, respectively. Thymidine 5′-diphosphate-Glc and UDP-galactose could replace UDP-Glc as the sugar nucleotide substrate to some extent, but the enzyme had no activity with NADP⁺. Soybean nodule UDP-Glc DH was labile in the absence of NAD⁺ and was inhibited by a heat-stable, low-molecular-mass solute in crude extracts of soybean nodules. UDP-Glc DH was also isolated from developing soybean seeds and shoots of 5-d-old wheat and canola seedlings and was shown to have similar affinities for UDP-Glc and NAD⁺ as those of the soybean nodule enzyme. UDP-Glc DH from all of these sources was most active in young, rapidly growing tissues.     

Effect of temperature on starch synthesis in potato tuber tissue and in amyloplasts

A sharp temperature optimum is observed at 21.5°C when the incorporation of [¹⁴C]sucrose into starch is measured with discs cut from developing tubers of potato (Solanum tuberosum L. cv Desirée). By contrast, increasing temperatures over the range 9 to 31°C only enhance release of ¹⁴C to respiratory CO₂ and incorporation of ¹⁴C into the ethanolsoluble fraction. By comparison, starch synthesis in discs from developing corms of cocoyam (Colocasia esculenta L. Schott) is increased by raising the temperature from 15 to 35°C. The significance of a relatively low temperature optimum for starch synthesis in potato is discussed in relation to the yield limitations imposed by continuously high soil temperatures. Amyloplasts isolated from protoplasts prepared from developing potato tubers contain activities of alkaline pyrophosphatase, NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, and phosphoglucomutase in addition to ADP-glucose-pyrophosphorylase, starch phosphorylase and starch synthase. Cell-free amyloplasts released by thinly slicing developing potato tubers synthesize starch from [¹⁴C]triose-phosphate generated from [¹⁴C]fructose-1,6-bisphosphate in the reaction medium. This starch synthesis is inhibited by addition of 10 millimolar inorganic phosphate and requires amyloplast integrity, suggesting the operation of a triose-phosphate/inorganic phosphate exchange carrier at the amyloplast membrane. The temperature optimum at 21.5°C observed with tissue discs is not observed with amyloplasts.     

Spinach leaf acetyl-coenzyme a synthetase: purification and characterization

Acetyl-coenzyme A (CoA) synthetase was purified 364-fold from leaves of spinach (Spinacia oleracea L.) using ammonium sulfate fractionation followed by ion exchange, dye-ligand, and gel permeation chromatography. The final specific activity was 2.77 units per milligram protein. The average M_r value of the native enzyme was about 73,000. The Michaelis constants determined for Mg-ATP, acetate, and coenzyme A were 150, 57, and 5 micromolar, respectively. The purified enzyme was sensitive to substrate inhibition by CoA with an apparent K_i for CoA of 700 micromolar. The enzyme was specific for acetate; other short and long chain fatty acids were ineffective as substrates. Several intermediates and end products of fatty acid synthesis were examined as potential inhibitors of acetyl-CoA synthetase activity, but none of the compounds tested significantly inhibited acetyl-CoA synthetase activity in vitro. The properties of the purified enzyme support the postulated role of acetyl-CoA synthetase as a primary source of chloroplast acetyl-CoA.     

Primer dependent and independent forms of soluble starch synthetase from developing barley endosperms

The activity of soluble starch synthetase (ADP-glucose: -1,4-glucan -4-glucosyltransferase) in the non-purified extract from 16 day-old Bomi barley endosperms (Hordeum vulgare L.) was low and the reaction was non-linear when plotted against protein concentration. Starch synthetase was purified by ammonium sulfate precipitation and DEAE-cellulose chromatography and separated into four fractions. In the absence of an added carbohydrate primer two of the four fractions catalized the synthesis of a methanol-precipitable -glucan when high concentrations of sodium citrate and bovine serum albumim were added. The rate of -glucan synthesis by the unprimed reaction was higher than for the primed reaction. The four enzyme fractions were active with ADP-Glc, but not with UDP-Glc, both in the primed and in the unprimed reaction.Abbreviations ADP-Glc adenosine diphosphate glucose - DEAE-cellulose diethylaminoethyl-cellulose - DDT dithiothreitol - EDTA ethylenediaminetetraacetic acid - GSH glutathione - kat katal (1 mol s^-1) - TRIS tris-(hydroxymethyl)-aminomethane - UDP-Glc uridine diphosphate glucose     

Photosynthetic Prokaryotes. Cell Differentiation and Functions : Edited by G.C. Papageorgiou and L. Packer Elsevier Biomedical; New York, 1983 405 pages. $65.00

A protein (designated as luffin) with an app. M_r of 26000, which inhibits protein synthesis in rabbit reticulocyte lysate, was purified to homogeneity from the seeds of Luffa cylindria roem by extraction with 20 mM Na phosphate buffer (pH 7.2) containing 0.2 M NaCl, ammonium sulfate fractionation, and chromatography on Sephacryl S-200 and CM-Sephadex C-25. Luffin exhibited 10-times as strong inhibitory activity against protein synthesis in rabbit reticulocyte lysate (IC₅₀, 0.42 ng/ml) as that of ricin A-chain, but it showed only a weak cytotoxicity against murine leukemia L1210 cells, an activity of 1/10⁶ to 1/10⁵ that of ricin.     

New actin-binding proteins from Dictyostelium discoideum

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca²⁺ -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [¹²⁵I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, α-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.     

Regulation of Starch Synthesis in the Bundle Sheath and Mesophyll of Zea mays L. : Intercellular Compartmentalization of Enzymes of Starch Metabolism and the Properties of the ADPglucose Pyrophosphorylases

The intercellular localization of enzymes involved in starch metabolism and the kinetic properties of ADPglucose pyrophosphorylase were studied in mesophyll protoplasts and bundle sheath strands separated by cellulase digestion of Zea mays L. leaves. Activities of starch synthase, branching enzyme, and ADPglucose pyrophosphorylase were higher in the bundle sheath, whereas the degradative enzymes, starch phosphorylase, and amylase were more evenly distributed and slightly higher in the mesophyll. ADPglucose pyrophosphorylase partially purified from the mesophyll and bundle sheath showed similar apparent affinities for Mg²⁺, ATP, and glucose-1-phosphate. The pH optimum of the bundle sheath enzyme (7.0-7.8) was lower than that of the mesophyll enzyme (7.8-8.2). The bundle sheath enzyme showed greater activation by 3-phosphoglycerate than did the mesophyll enzyme, and also showed somewhat higher apparent affinity for 3-phosphoglycerate and lower apparent affinity for the inhibitor, orthophosphate. The observed activities of starch metabolism pathway enzymes and the allosteric properties of the ADPglucose pyrophosphorylases appear to favor the synthesis of starch in the bundle sheath while restricting it in the mesophyll.     

Control of starch and exocellular polysaccharides biosynthesis by gibberellic acid with cells of sweet potato cultured in vitro

The regulation of starch synthesis and exocellular polysaccharide synthesis by GA₃ was studied with cells of sweet potato grown as suspension in glycerol medium. In the presence of GA₃, and under normal cell growth, starch formation was inhibited. The incorporation activity (starch synthesis) from ADP-[¹⁴C] glucose or UDP-[¹⁴C] glucose with GA₃ treated cells was reduced. On the other hand, the synthesis of exocellular polysaccharides composed of glucose, galactose, mannose and arabinose etc., was stimulated and a clear increase of the Man/Ara ratio was observed in the presence of GA₃. These results may indicate that GA₃ affects the regulation of starch synthesis and exocellular polysaccharide synthesis.     

Statistically designed optimisation of enzyme catalysed starch removal from potato pulp

Potato pulp is a high-volume, low-value byproduct stream resulting from the industrial manufacture of potato starch. The pulp is a rich source of biologically functional dietary fibers, but the targeted valorisation of the fibers requires removal of the residual starch from the pulp. The objective of this study was to release the residual starch, making up 21–22% by weight of the dry matter, from the potato pulp in a rational way employing as few steps, as few enzyme activities, as low enzyme dosages, as low energy input (temperature and time), and as high pulp dry matter as possible. Starch removal to obtain dietary fibers is usually accomplished via a three step, sequential enzymatic treatment procedure using a heat stable α-amylase, protease, and amyloglucosidase. Statistically designed experiments were performed to investigate the influence of enzyme dose, amount of dry matter, incubation time and temperature on the amount of starch released from the potato pulp. The data demonstrated that all the starch could be released from potato pulp in one step when 8% (w/w) dry potato pulp was treated with 0.2% (v/w) (enzyme/substrate (E/S)) of a thermostable Bacillus licheniformis α-amylase (Termamyl^® SC) at 70 °C for at least 65 min. The study also indicated that the amount of other carbohydrates released from the pulp during the release of starch was less than using the AOAC Official Method 985.29 and another recently published starch release method employed as a pretreatment for enzymatic upgrading of a pectinaceous potato pulp fiber.     

Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major

Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential.     

A sucrose non-fermenting-1-related protein kinase 1 gene from potato, <Emphasis Type="Italic">StSnRK1</Emphasis>, regulates carbohydrate metabolism in transgenic tobacco

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been shown to play an essential role in regulating saccharide metabolism and starch biosynthesis of plant. The regulatory role of StSnRK1 from potato in regulating carbohydrate metabolism and starch accumulation has not been investigated. In this work, a cDNA encoding the SnRK1 protein, named StSnRK1, was isolated from potato. The open reading frame contained 1545 nucleotides encoding 514 amino acids. Subcellular localization analysis in onion epidermal cells indicated that StSnRK1 protein was localized to the nucleus. The coding region of StSnRK1 was cloned into a binary vector under the control of 35S promoter and then transformed into tobacco to obtain transgenic plants. Transgenic tobacco plants expressing StSnRK1 were shown to have a significant increased accumulation of starch content, as well as sucrose, glucose and fructose content. Real-time quantitative PCR analysis indicated that overexpression of StSnRK1 up-regulated the expression of sucrose synthase (NtSUS), ADP-glucose pyrophosphorylase (NtAGPase) and soluble starch synthase (NtSSS III) genes involved in starch biosynthesis in the transgenic plants. In contrast, the expression of sucrose phosphate synthase (NtSPS) gene was decreased in the transgenic plants. Meanwhile, enzymatic analyses indicated that the activities of major enzymes (SUS, AGPase and SSS) involved in the starch biosynthesis were enhanced, whereas SPS activity was decreased in the transgenic plants compared to the wild-type. These results suggest that the manipulation of StSnRK1 expression might be used for improving quality of plants in the future.