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Preparations enriched in plastics were used to investigate the location of ADP-glucose pyrophosphorylase (AGPase) in the developing endosperm of maize (Zea mays L.). These preparations contained more than 25% of the total activity of the plastic marker enzymes alkaline pyrophosphatase and soluble starch synthase, less than 2% of the cytosolic marker enzymes alcohol dehydrogenase and pyrophosphate, fructose 6-phosphate 1-phosphotransferase, and approximately 3% of the AGPase activity. Comparison with the marker enzyme distribution suggests that more than 95% of the activity of AGPase in maize endosperm is extra-plastidial. Two proteins were recognized by antibodies to the small subunit of AGPase from maize endosperm Brittle-2 (Bt2). The larger of the two proteins was the major small subunit in homogenates of maize endosperm, and the smaller, less abundant of the two proteins was enriched in preparations containing plastics. These results suggest that there are distinct plastidial and cytosolic forms of AGPase, which are composed of different subunits. Consistent with this was the finding that the bt2 mutation specifically eliminated the extra-plastidial AGPase activity and the larger of the two proteins recognized by the antibody to the Bt2 subunit

The aim of this work was to characterise the metabolism of starch in developing embryos of oilseed rape (Brassica napus L. cv. Topaz). The accumulation of starch in embryos in siliques which were darkened or had been exposed to the light was similar, suggesting that the starch is synthesised from imported sucrose rather than via photosynthesis in the embryo. Starch content and the activities of plastidial enzymes required for synthesis of starch from glucose 6-phosphate (Glc6P) both peaked during the early-mid stage of cotyledon development (i.e. during the early part of oil accumulation) and then declined. The mature embryo contained almost no starch. The starch-degrading enzymes α-(EC 3.2.1.1) and β-amylase (EC 3.2.1.2) and phosphorylase (EC 2.4.1.1) were present throughout development. Most of the activity of these three enzymes was extraplastidial and therefore unlikely to be involved in starch degradation, but there were distinct plastidial and extraplastidial isoforms of all three enzymes. Activity gels indicated that distinct plastidial isoforms increase during the change from net synthesis to net degradation of starch. Plastids isolated from embryos at stages both before and after the maximum starch content could convert Glc6P to starch although the rate was lower at the later stage. The results are consistent with the idea that starch synthesis and degradation occur simultaneously during embryo development. The possible roles of transient starch accumulation during embryo development are discussed.

Rumen bacterium Pseudobutyrivibrio ruminis strain k3 utilized over 90 % sucrose added to the growth medium as a sole carbon source. Zymographic studies of the bacterial cell extract revealed the presence of a single enzyme involved in sucrose digestion. Thin layer chromatography showed fructose and glucose-1-phosphate (Glc1P) as end products of the digestion of sucrose by identified enzyme. The activity of the enzyme depended on the presence of inorganic phosphate and was the highest at the concentration of phosphate 56 mmol/L. The enzyme was identified as the sucrose phosphorylase of molar mass approx. 54 kDa and maximum activity at pH 6.0 and 45 deg C. The calculated Michaelis constants for Glc1P formation and release of fructose by partially purified enzyme were 4.4 and 8.56 mmol/L while the maximum velocities of the reaction were 1.19 and 0.64 micromol/L per mg protein per min, respectively.

We have demonstrated that expression of genes involved in starch and storage protein synthesis of the maize (Zea mays L.) endosperm are coordinated. Genetic lesions altering synthetic events in one biosynthetic pathway affect expression of genes in both pathways. Initial studies focused on shrunken2 (sh2) and brittle2 (bt2) mutants because these genes encode subunits of the same enzyme, ADP-glucose pyrophosphorylase. Analysis of various sh2- and bt2- mutant alleles showed that the most severe mutations also conditioned the largest increase in transcripts. The analysis was extended by monitoring the transcripts of the genes, shrunken 1 (sh1, structural gene for Suc synthase), sh2, bt2, waxy1 (wx1, structural gene for starch synthase), and those of the large and small zeins in isogenic maize lines at 14, 22, and 30 d postpollination. Endosperms were wild type for all of these genes or contained sh1-, sh2-, bt1-, bt2-, opaque2 (o2-), or amylose-extender1 (ae1-) dull1 (du1-) wx1- mutations. Transcripts increased continually throughout kernel development in the mutants relative to the standard W64A used. Variation in the amount of Suc entering the developing seed also altered transcript amounts. The results indicate that starch and protein biosynthetic genes act in a concerted manner, and both are sensitive to mutationally induced differences

Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 micromole of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4 degrees C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP

Lysine (Lys)-195 in the homotetrameric ADP-glucose pyrophosphorylase (ADPGlc PPase) from Escherichia coli was shown previously to be involved in the binding of the substrate glucose-1-phosphate (Glc-1-P). This residue is highly conserved in the ADPGlc PPase family. Site-directed mutagenesis was used to investigate the function of this conserved Lys residue in the large and small subunits of the heterotetrameric potato (Solanum tuberosum) tuber enzyme. The apparent affinity for Glc-1-P of the wild-type enzyme decreased 135- to 550-fold by changing Lys-198 of the small subunit to arginine, alanine, or glutamic acid, suggesting that both the charge and the size of this residue influence Glc-1-P binding. These mutations had little effect on the kinetic constants for the other substrates (ATP and Mg2+ or ADP-Glc and inorganic phosphate), activator (3-phosphoglycerate), inhibitor (inorganic phosphate), or on the thermal stability. Mutagenesis of the corresponding Lys (Lys-213) in the large subunit had no effect on the apparent affinity for Glc-1-P by substitution with arginine, alanine, or glutamic acid. A double mutant, S(K198R)L(K213R), was also obtained that had a 100-fold reduction of the apparent affinity for Glc-1-P. The data indicate that Lys-198 in the small subunit is directly involved in the binding of Glc-1-P, whereas they appear to exclude a direct role of Lys-213 in the large subunit in the interaction with this substrate

The expression of the enzyme UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) from potato (Solanum tuberosum L.) was analysed with respect to sink-source interactions and potato tuber storage. The highest level of expression was found in developing tubers, the strongest sink tissue. Storage of mature tubers at low temperatures led to an increase of the steady-state level of UGPase mRNA, implicating a role of this enzyme in the process of \"cold-sweetening\". Transgenic plants were created expressing UGPase antisensee RNA under the control of the 35S promoter of the Cauliflower Mosaic Virus with the polyadenylation signal of the octopine-synthase gene. Regenerated plants were tested for reduction of UGPase at the RNA, protein and activity levels. Plants with a 95%—96% reduction of UGPase activity in growing tubers showed no change in growth and development. Also, carbohydrate metabolism in tubers of these plants was not substantially affected, indicating that only 4% of the wild-type UGPase activity is sufficient for the enzyme to function in plant growth and development.

The intracellular location of ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato (Lycopersicon esculentum Mill) has been investigated by immunolocalization. With the use of a highly specific anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm as well as plastids at both the light and electron microscope levels. The immunogold particles in plastids were localized in the stroma and at the surface of the starch granule, whereas those in the cytoplasm occurred in cluster-like patterns. Contrary to the fruit, the labeling in tomato leaf cells occurred exclusively in the chloroplasts. These data demonstrate that AGP is localized to both the cytoplasm and plastids in developing pericarp cells of tomato.

We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

Previous experiments have shown that carbohydrate partitioning in leaves of potato (Solanum tuberosum L.) plants can be modified by antisense repression of the triose phosphate translocator (TPT), favoring starch accumulation during the light period, or by leaf-specific antisense repression of ADP-glucose pyrophosphorylase (AGPase), reducing leaf starch content. These experiments showed that starch and sucrose synthesis can partially replace each other. To determine how leaf metabolism acclimates to an inhibition of both pathways, transgenic potato (S. tuberosum L. cv Desiree) plants, with a 30% reduction of the TPT achieved by antisense repression, were transformed with an antisense cDNA of the small subunit of AGPase, driven by the leaf-specific ST-LS1 promoter. These double-transformed plants were analyzed with respect to their carbohydrate metabolism, and starch accumulation was reduced in all lines of these plants. In one line with a 50% reduction of AGPase activity, the rate of CO2 assimilation was unaltered. In these plants the stromal level of triose phosphate was increased, enabling a high rate of triose phosphate export in spite of the reduction of the TPT protein by antisense repression. In a second line with a 95% reduction of AGPase activity, the amount of chlorophyll was significantly reduced as a consequence of the lowered triose phosphate utilization capacity