Purification and properties of adenosine diphosphoglucose pyrophosphorylase from sweet corn

A 40-fold purification of adenosine diphosphoglucose pyrophosphorylase from sweet corn (Zea mays var. Golden Beauty) revealed the enzyme to be specific for adenosine triphosphate. The enzyme has an absolute requirement for Mg²⁺ and is activated by 3-phosphoglycerate and to a lesser extent by ribose-5-phosphate and fructose-6-phosphate. The apparent Km values of the enzyme for glucose-1-phosphate, adenosine triphosphate, pyrophosphate, and adenosine diphosphoglucose are 1.9 × 10⁻⁴, 3.2 × 10⁻⁵, 3.3 × 10⁻⁵, and 6.2 × 10⁻⁴m, respectively. Pyrophosphate inhibits adenosine diphosphoglucose synthesis competitively (Ki = 3.8 × 10⁻⁷m), while orthophosphate and sulfate appear to inhibit the reacion noncompetitively. These results show that the production of this sugar nucleotide can be controlled by the concentration of pyrophosphate.     

Nucleotide sequence and expression analysis of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene.

The nucleotide sequence of the Acetobacter xylinum uridine diphosphoglucose pyrophosphorylase gene was determined; this is the first procaryotic uridine diphosphoglucose pyrophosphorylase gene sequence reported. The sequence data indicated that the gene product consists of 284 amino acids. This finding was consistent with the results obtained by expression analysis in vivo and in vitro in Escherichia coli.     

Biosynthesis of monoterpenes. Partial purfication and characterization of a bicyclic monoterpenol dehydrogenase from sage (Salvia officinalis)

Galactose 1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose 1-phosphate uridylyltransferase, EC 2.7.7.12) was isolated from human red cells by DEAE-cellulose and hydroxylapatite chromatography. The enzyme consists. of two similar subunits of molecular weight 44,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular weight of the enzyme was found to be 67,000 by Sephadex G-200 chromatography and 88,000 by ultracentrifugation studies in sucrose density gradients. The specific activity of the purified enzyme was about 40 μmoles per min per mg of protein.     

Adenosine diphosphoglucose-starch glucosyltransferases from developing kernels of waxy maize

Two adenosine diphosphoglucose: α-1,4-glucan α-4-glucosyl-transferases were extracted from kernels of waxy maize harvested 22 days after pollination and separated by gradient elution from a diethylaminoethyl-cellulose column. Both fractions could utilize amylopectin, amylose, glycogen, maltotriose and maltose as primers. The rate of glucose transfer from adenosine diphosphoglucose to rabbit liver glycogen of fraction II was 78% of the rate of glucose transfer to amylopectin, but with fraction I the rate of transfer of glucose to rabbit liver glycogen was 380% of that observed to amylopectin. Glucan synthesis in the absence of added primer was found in fraction I in the presence of 0.5 m sodium citrate and bovine serum albumin. The unprimed product was a methanol-precipitable glucan with principally α-1,4 linkages and some α-1,6 linkages, and its iodine spectrum was similar to that of amylopectin.     

Developmental regulation of multiple forms of UDPglucose pyrophosphorylase of Dictyostelium

Uridine diphosphoglucose pyrophosphorylase (UTP:α-d-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) is a developmentally regulated enzyme in Dictyostelium discoideum essential for the completion of its life cycle. During vegetative growth and the early stages of differentiation the specific activity of the enzyme remains constant. However, it increases threefold by the time fruiting bodies are formed. We have identified a developmentally specific form of uridine diphosphoglucose pyrophosphorylase, altered in both isoelectric point and apparent molecular weight, by resolving crude extracts of cells on two-dimensional denaturing polyacrylamide gels, renaturing the protein in situ, and localizing active enzyme with a histochemical stain. Quantitation of the amount of enzyme stain deposited in the gels shows that the activity in the new form can account for the increase observed in development. The appearance of the developmental form of the enzyme requires de novo protein synthesis since it is inhibited by cycloheximide. Immunoprecipitation of uridine diphosphoglucose pyrophosphorylase from in vivo and in vitro synthesized proteins has revealed heterogeneity not previously detected in the enzyme from both vegetative and developed cells. Two different proteins are synthesized in vitro by mRNA from either vegetative or developed cells. These two proteins are also found in vivo in developed cells. Only one of the two proteins is found in vegetative cells. Enzyme protein synthesized in vivo appears to be modified after translation. Therefore, the observed heterogeneity in uridine diphosphoglucose pyrophosphorylase found in vivo appears due both to post-translational modification and to synthesis of two polypeptides from one or more species of mRNA.     

Uridine diphosphoglucose dehydrogenase in the intervertebral disc

Synopsis Intervertebral discs of an old sheep and a young pig were examined for the presence of cells containing the enzyme uridine diphosphoglucose dehydrogenase. In the sheep, the inner anulus had a higher proportion of active cells than the outer anulus; in the pig, there was no difference. From a consideration of cell numbers, it is suggested that there is an accumulation of glycosaminoglycans in the centre of the disc rather than an increased production rate. Notochordal cells in the pig disc contain uridine diphosphoglucose dehydrogenase and are capable of producing glycosaminoglycans.     

Inhibition by Actinomycin D of Uridine Diphosphoglucose Synthetase Activity During Differentiation of Dictyostelium discoideum

Actinomycin D inhibits the increase in specific activity of uridine diphosphoglucose synthetase during differentiation of the cellular slime mold, Dictyostelium discoideum. The degree to which this inhibition occurs is strictly correlated with, and may result from, the general inhibition of morphogenesis by actinomycin D.     

Partial purification and characterization of glycogen phosphorylase from Dictyostelium discoideum

Glycogen phosphorylase was isolated from cells of Dictyostelium discoideum in the culmination stage of development and purified 35-fold. The enzyme had a pH optimum of 6.9 and contained sulfhydryl groups essential for activity. The K(m) values for phosphate and glycogen were 3 mm and 0.06% (w/v), respectively. No dependence on, or stimulation by, any nucleotide was observed and a wide variety of nucleotides and glycolytic intermediates did not inhibit the enzyme. Nucleotide sugars competitively inhibited the enzyme. Guanosine diphosphoglucose and adenosine diphosphoglucose were the most effective, and uridine diphosphoglucose was the least effective of the nucleotide sugars tested. The specific activity of glycogen phosphorylase increased from about 0.004 unit per mg of protein in aggregating cells to about 0.024 unit per mg in culminating cells, and then decreased during sorocarp formation. This increase in enzyme specific activity during the starvation and aging of the system can account for the increased rate of glycogen degradation during this period of development. Amylase specific activity, measured at pH 4.8 and 6.9, varied between 0.005 and 0.013 unit per mg of protein during all stages of development.     

Human galactose-1-phosphate uridylyltrsferase: purification and comparison of the red blood cell and placental enzymes

Galactose-1-phosphate Uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate Uridylyltransferase, EC 2.7.7.12) has been purified from human red blood cells and placental tissue. The placental enzyme was obtained as a homogeneous protein with a specific activity of about 100 units/mg of protein by a combination of previously published methods (G. R. Helmer, Jr., and V. P. Williams, 1981,Arch. Biochem. Biophys.210, 573–580) and concanavalin A-Sepharose chromatography. The properties of the two enzyme forms have been examined with respect to subunit size, electrophoretic properties, isozyme distribution, kinetic patterns, and immunological properties.     

A unique adenosine diphosphoglucose pyrophosphorylase associated with maize embryo tissue

A comparison of heat stabilities and various kinetic properties between the adenosine diphosphoglucose pyrophosphorylases isolated from endosperm and embryo tissues from starchy maize seeds indicates that the adenosine diphosphoglucose pyrophosphorylase associated with the embryo is distinct from the enzyme isolated from the endosperm. The embryo enzyme is more stable to incubation for 5 minutes at 60 C while the endosperm enzyme is labile to this treatment. Both enzymes are activated by glycerate-3-P. The embryo enzyme is more sensitive to inhibition by phosphate than is the endosperm enzyme. Glycerate-3-P, which reverses the inhibition of the endosperm enzyme by phosphate, has little effect on the phosphate inhibition of the embryo enzyme. Other kinetic studies distinguish the two enzymes.     

The role of the skin of Rana pipiens in sulfate excretion

• 1.1. The intact whole skin of Rana pipiens excretes sulfate and this excretion is increased by sulfate loading of the animal,
• 2.2. Ninety-five percent of the excretion of sulfate seen following sulfate loading is via the skin, while a small amount is handled by the urinary system.

     

T-Even Bacteriophage-Tolerant Mutants of Escherichia coli B III. Nature of the tet Defect

T-even phage-tolerant (tet) mutants of Escherichia coli B are shown to lack the enzyme uridine diphosphoglucose pyrophosphorylase and thus produce nonglucosylated progeny phage deoxyribonucleic acid.     

The glutathione dependence of inorganic sulfate formation from l- or d-cysteine in isolated rat hepatocytes

The GSH dependence of the metabolic pathways involved in the conversion of cysteine to sulfate in intact cells has been investigated. It was found that hepatocyte-catalysed sulfate formation from added

Full-size image

-cysteine did not occur if hepatocyte GSH was depleted beforehand, but was restored when GSH levels recovered. Furthermore, sulfate formation did not recover in GSH-depleted hepatocytes if GSH synthesis was prevented with buthionine sulfoximine. Thiosulfate formation was, however, markedly enhanced in GSH-depleted hepatocytes. These results suggest that thiosulfate is an intermediate in the formation of inorganic sulfate from

Full-size image

-cysteine and that GSH was required for the conversion of thiosulfate to inorganic sulfate. Much less sulfate was formed if the cysteine was replaced with cysteinesulfinate. Furthermore, sulfate formation from

Full-size image

-cysteine was markedly inhibited by the addition of the transaminase inhibitor

Full-size image

-cycloserine or the γ-cystathionase inhibitor

Full-size image

-propargylglycine. The major routes of sulfate formation from

Full-size image

-cysteine therefore seems to involve pathways that do not involve

Full-size image

-cysteinesulfinate. Similar amounts of sulfate were formed from

Full-size image

-cysteine as

Full-size image

-cysteine. Thiosulfate instead of sulfate was also formed in GSH-depleted hepatocytes. However, sulfate formation from

Full-size image

-cysteine differed from

Full-size image

-cysteine in that it was inhibited by the

Full-size image

-aminoacid oxidase inhibitor sodium benzoate and was not affected by transaminase or γ-cystathionase inhibitors. These results suggest that thiosulfate is an intermediate in sulfate formation from

Full-size image

-cysteine and involves the oxidation of

Full-size image

-cysteine by

Full-size image

-amino acid oxidase to form β-mercaptopyruvate.     

Stability in vitro of uridine diphosphoglucose pyrophosphorylase in Dictyostelium discoideum

The stability of uridine diphosphoglucose pyrophosphorylase was examined in extracts prepared at different stages of development in Dictyostelium discoideum. In the early stages, the kinetics of inactivation were nonlinear, and, therefore, it was not possible to determine the specific enzyme activity. In the later stages of development, the enzyme was stable, but it could be rapidly inactivated by a heat-labile inhibitor present in extracts prepared at an early stage.     

Sterol glucosylation by the plasma membrane from cotyledons of Phaseolus aureus

Membrane fractions were isolated from dark grown cotyledons of Phaseolus auneus by differential and sucrose density gradient centrifugation. Endoplasmic reticulum-, Golgi apparatus- and plasma membrane-rich fractions were identified by their respective enzymic activities and tested for their ability to transfer glucose from UDP-glucose to endogenous sterols to form steryl glucosides. The glucosyltransferase activity was shown to be located mainly at the plasma membrane.ABBREVIATIONS SG steryl glucoside - ASG acylated steryl glucoside - UDP-glc Uridine diphosphoglucose     

Effect of ferric sulfate on activity of moderately thermophilic acidophilic iron-oxidizing microorganisms

The effect of high ferric sulfate concentrations on the organisms predominating in biohydrometallurgical processes (bacteria of genus Sulfobaсillus and archaea of the genus Acidiplasma) was studied. Ability of the studied strains to grow and oxidize ferrous iron in the media with 125 to 500 mM ferric sulfate was determined. High concentrations of ferric sulfate significantly inhibited the oxidative activity and growth of the studied microorganisms. Bacteria of the genus Sulfobaсillus were found to be incapable of active iron oxidation in the presence of ferric iron sulfate at concentrations exceeding 250 mM. Archaea of the genus Acidiplasma oxidized ferrous iron completely in the presence of 500 mM Fe³⁺. Microbial growth was suppressed by relatively low ferric sulfate concentrations. Almost no growth occurred at ferric sulfate concentrations exceeding 199 mM, while lysis of the cells of all studied strains was observed at higher Fe³⁺ concentrations. Archaea (genus Acidiplasma, family Ferroplasmaceae) were shown to be more tolerant to high ferric sulfate concentrations than bacteria of the genus Sulfobaсillus. The results obtained may be used for improvement of biohydrometallurgical technologies and are also important for the understanding of the patterns of formation of microbial communities carrying out the technological processes.     

Regulation of sulfate transport in filamentous fungi

Inorganic sulfate enters the mycelia of Aspergillus nidulans, Penicillium chrysogenum, and Penicillium notatum by a temperature-, energy-, pH-, ionic strength-, and concentration-dependent transport system (“permease”). Transport is unidirectional. In the presence of excess external sulfate, ATP sulfurylase-negative mutants will accumulate inorganic sulfate intracellularly to a level of about 0.04 m. The intracellular sulfate can be retained against a concentration gradient. Retention is not energy-dependent, nor is there any exchange between intracellular (accumulated) and extracellular sulfate. The sulfate permease is under metabolic control. Sulfur starvation of high methionine-grown mycelia results in about a 1000-fold increase in the specific sulfate transport activity at low external sulfate concentrations. l-Methionine is a metabolic repressor of the sulfate permease, while intracellular sulfate and possibly l-cysteine (or a derivative of l-cysteine) are feedback inhibitors. Sulfate transport follows hyperbolic saturation kinetics with a Michaelis constant (Km) value of 6 × 10⁻⁵ to 10⁻⁴m and a V_max (for maximally sulfurstarved mycelia) of about 5 micromoles per gram per minute. Refeeding sulfur-starved mycelia with sulfate or cysteine results in about a 10-fold decrease in the V_max value with no marked change in the Km. Azide and dinitrophenol also reduce the V_max.     

Expression of extracellular polysaccharides and proteins by clinical isolates of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in response to environmental conditions

The opportunistic pathogen Pseudomonas aeruginosa causes chronic respiratory infections in patients with cystic fibrosis (CF). Persistence of this bacterium is attributed to its ability to form biofilms which rely on an extracellular polymeric substance matrix. Extracellular polysaccharides (EPS) and secreted proteins are key matrix components of P. aeruginosa biofilms. Recently, nebulized magnesium sulfate has been reported as a significant bronchodilator for asthmatic patients including CF. However, the impact of magnesium sulfate on the virulence effect of P. aeruginosa is lacking. In this report, we investigated the influence of magnesium sulfate and other environmental factors on the synthesis of alginate and secretion of proteins by a mucoid and a non-mucoid strain of P. aeruginosa, respectively. By applying the Plackett-Burman and Box-Behnken experimental designs, we found that phosphates (6.0 g/l), ammonium sulfate (4.0 g/l), and trace elements (0.6 mg/l) markedly supported alginate production by the mucoid strain. However, ferrous sulfate (0.3 mg/l), magnesium sulfate (0.02 g/l), and phosphates (6.0 g/l) reinforced the secretion of proteins by the non-mucoid strain.