Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45–60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD⁺, NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20–50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.     

AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides

The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer.     

Proteomic analysis of curdlan-producing Agrobacterium sp. in response to pH downshift

During batch cultivation of Agrobacterium sp. ATCC 31750, proteome analysis in response to a pH downshift from 7.0 to 5.5 was carried out using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. When the pH of the exponentially growing Agrobacterium sp. culture was downshifted to pH 5.5, the synthesis level of 27 intracellular proteins showed significant changes in level over a prolonged period of time compared with the batch culture controlled at pH 7.0. In particular, the intracellular protein level of the beta-1,3-glucan synthase catalytic subunit, UTP-glucose-1-phosphate uridylyltransferase, and phosphoglucomutase, which are key metabolic enzymes in the curdlan biosynthesis pathway, were more than 10-, 3- and 17-times higher in the low pH culture. On the other hand, the level of orotidine5-phosphate decarboxylase (conversion of OMP to UMP) was significantly up-regulated after pH downshift. The accumulation of UMP may direct the metabolic flow towards the biosynthetic route of UTP, which is a key metabolic precursor for UDP-glucose. Therefore, it is possible that increase of cellular metabolic enzymes during pH downshift culture can enhance the metabolic flux of the biosynthesis of key precursor, such as UTP- and UDP-glucose, resulting in an increase in curdlan biosynthesis.     

The composition of soluble nucleotides in the developing wheat grain

A method is described for the extraction and measurement of soluble nucleotides from wheat grain. Nucleotides were separated (80-90% recovery) by paper chromatography followed by electrophoresis. The nucleotides extracted were ADP-glucose, ATP, ADP, AMP, and NAD; UDP-glucose, UTP, UDP, and UMP with smaller quantities of cytidine nucleotides.     

Nucleotide pools of growing,synchronized and stressed cultures of Saccharomyces cerevisiae

High pressure liquid chromatography has been used to study the acid soluble nucleotide pool of Saccharomyces cerevisiae under different conditions of growth. ATP, ADP, AMP, NAD, GTP, UTP, UDP, CTP, CDP, and UDP-sugars plus UMP could be separated and were found in concentrations higher than 0.1 mumol per g yeast cell dry weight (= detection limit). During glucose-limited continuous culture the levels of individual nucleotides depended on the growth rate, which was most pronounced with pyrimidine (uridine, cytidine) nucleotides. The energy charge (E.C.) remained high (0.9) at all growth rates (0.07-0.3 h-1). During synchronized growth at a constant growth rate (0.11 h-1) almost all nucleotide levels and the E.C. remained at constant values with the only exception of UDP-sugars and UMP of which increased levels were found during the phase of budding. Under conditions of metabolic stress (addition of antimycin A, deoxyglucose plus iodoacetate) pronounced changes in the levels of purine (adenine and guanine) nucleotides and the E.C. were observed. All other nucleotides were less influenced by these conditions. Only under these conditions IMP accumulation was observed. The results strongly argue against the significance of purine nucleotide or E.C. measurements under viable conditions. In contrast, changes in the levels of pyrimidine nucleotides seem to be indicative of changes in the flux through the metabolic pathways where they act as coenzymes.     

The role of nucleoside diphosphatase in a uridine nucleotide cycle associated with lactose synthesis in rat mammary-gland Golgi apparatus.

1. UDP-galactose utilization by isolated Golgi vesicles or rat mammary gland synthesizing lactose causes accumulation of UMP but not UDP, although UDP is the immediate product of lactose synthase (EC 2.4.1.22). 2. This can be ascribed to a nucleoside diphosphatase (EC 3.6.1.6), specific for UDP, GDP and IDP, activated by bivalent metal ions and apparently located on the luminal face of the Golgi membrane. 3. The uridine diphosphatase activity exceeds the total galactosyltransferase activity 5-fold, and is estimated to maintain UDP at about 14 micrometer within the Golgi lumen. 4. Evidence is given that UMP, but not UDP, penetrates the membrane and that UMP is rephosphorylated to UDP by a UMP kinase located in the cytosol. 5. Golgi-cytosol relationships with respect to lactose synthesis are formulated in terms of a uridine nucleotide cycle which throws new light on the energy cost and possible regulation of lactose synthesis.     

Labeling of precursor pools for glycosphingolipid biosynthesis. Incorporation of [3H]galactose by rat hepatocytes in primary culture

UDP-galactose and UDP-glucose are the immediate sources of monosaccharide residues in glycosphingolipid biosynthesis. The incorporation of [6-3H]D-galactose into these compounds was measured in primary cultures of rat hepatocytes, which take up and metabolize galactose rapidly. The UDP-glucose and UDP-galactose content of hepatocytes, determined enzymatically and by the HPLC-analysis of UDP-sugars, was 1.87 +/- 0.22 and 0.51 +/- 0.06 nmol/mg protein, respectively. Galactose concentrations in the medium of up to 7.5 microM did not influence the intracellular levels of UDP-glucose and UDP-galactose. Although the specific radioactivity of these precursor pools did not reach a constant plateau, conditions were defined that allow the calculation of rates of glycolipid synthesis from added labeled galactose. They include the replacement of glucose in the culture medium by sodium pyruvate and D-galactose.     

Uridine diphosphoglucose pyrophosphorylase activity and differentiation in the cellular slime mold Physarum polycephaluno

The specific activity of uridine 5'-triphosphate:alpha-d-glucose 1-phosphate uridyltransferase (EC 2.7.7.9) (also called uridine 5'-diphosphate [UDP]-glucose pyrophosphorylase) has been found to increase up to eightfold during spherule formation by the slime mold Physarum polycephalum. The enzyme accumulates during the first 8 to 9 h after initiation of spherule formation, declines to basal levels found in vegetative microplasmodia by 15 h, and is undetectable in completed spherules. Specific activities for UDP-glucose pyrophosphorylase in vegetative microplasmodia range from 15 to 30 nmol of UDP-glucose formed per min per mg of protein, whereas accumulated levels during spherule formation can attain a specific activity as high as 125 nmol of UDP-glucose formed per min per mg of protein. The scheduling and extent of accumulation are critically dependent on an early log-phase age of microplasmodia originally induced to form spherules. Spherule induction by 0.2 M or 0.5 M mannitol delays this schedule in a variable and unpredictable manner. Spherule-forming microplasmodia which have accumulated high levels of UDP-glucose pyrophosphorylase spontaneously excrete the enzyme when transferred to salts medium containing 0.2 M or 0.5 M mannitol. The excreted enzyme is subsequently destroyed or inactivated. Studies with preferential inhibitors of macromolecular synthesis indicate that accumulation of UDP-glucose pyrophosphorylase requires concomitant protein synthesis and prior ribonucleic acid synthesis.     

Pathways of purine metabolism in human adipocytes. Further evidence against a role of adenosine as an endogenous regulator of human fat cell function

Previous results demonstrated that the adenosine that accumulates in human fat cell suspensions is derived from extracellular sources (Kather, H. (1988) J. Biol. Chem. 263, 8803-8809). To get insight into the mechanisms responsible for the lack of adenosine release, extracellular adenine nucleotide catabolism was minimized by 10 mmol/liter beta-glycerophosphate and 10 mumol/liter alpha,beta-methyleneadenosine 5'-diphosphate. Intracellular adenine nucleotide catabolism resulted in a release of inosine and hypoxanthine under these conditions that was increased markedly by isoproterenol. Experiments with inhibitors of adenosine deaminase and adenosine kinase indicated that the production of inosine and hypoxanthine proceeded via AMP deamination. Consistently, IMP levels were increased transiently in the presence of isoproterenol. In addition, the cells possessed a nucleotide phosphomonoesterase that was resistant to the inhibitory actions of ATP and alpha,beta-methyleneadenosine 5'-diphosphate and showed preference for IMP over AMP. Adenosine (approximately 1 nmol/10(6) cells/h) was also produced inside the cells. However, adenosine production was unrelated to ATP turnover via adenylate cyclase, and any adenosine formed was immediately reconverted to adenine nucleotides in the absence and presence of isoproterenol. It was concluded that adenosine is not released by intact human adipocytes, because the alternative routes of intracellular AMP catabolism are compartmentalized (at least in functional terms), and adenosine kinase is not saturated with substrate in the absence and presence of isoproterenol.     

Extracellular metabolism of nucleotides in neuroblastoma x glioma NG108-15 cells determined by capillary electrophoresis

1. The metabolism of extracellular nucleotides in NG108-15 cells, a neuroblastoma × glioma hybrid cell line, was studied by means of capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC).2. In NG108-15 cells ATP, ADP, AMP, UTP, UDP, and UMP were hydrolyzed to the nucleosides adenosine and uridine indicating the presence of ecto-nucleotidases and ecto-phosphatases. The hydrolysis of the purine nucleotides ATP and ADP was significantly faster than the hydrolysis of the pyrimidine nucleotides UTP and UDP.3. ATP and UTP breakdown appeared to be mainly due to an ecto-nucleotide- diphosphohydrolase. ADP, but not UDP, was initially also phosphorylated to some extent to the corresponding triphosphate, indicating the presence of an adenylate kinase on NG108-15 cells. The alkaline phosphatase (ALP) inhibitor levamisole did not only inhibit the hydrolysis of AMP to adenosine and of UMP to uridine, but also the degradation of ADP and to a larger extent that of UDP. ATP and UTP degradation was only slightly inhibited by levamisole.4. These results underscore the important role of ecto-alkaline phosphatase in the metabolism of adenine as well as uracil nucleotides in NG108-15 cells. Dipyridamole, a potent inhibitor of nucleotide breakdown in superior cervical ganglion cells, had no effect on nucleotide degradation in NG108-15 cells.5. Dipyridamole, which is a therapeutically used nucleoside reuptake inhibitor in humans, reduced the extracellular adenosine accumulation possibly by allosteric enhancement of adenosine reuptake into the cells.     

Mechanism of Apparent Transcription Inhibition by Methyl Mercury in Cerebellar Neurons

Abstract: We have investigated the mechanism of inhibition of RNA synthesis by methyl mercury (MeHg) in isolated neonatal rat cerebellar cells. Each of the three component steps involved in the incorporation of exogenous [³H]uridine into cellular RNA was examined separately in whole-cell and/or subcellular preparations. Nuclear RNA polymerase activity was measured in preparations containing both free nuclei and whole cells. Incorporation of [³H]UTP into nuclear RNA was found to be unimpaired at concentrations of MeHg that inhibited whole-cell incorporation of [³H]uridine by > 75%. Cellular uptake of [³H]uridine was assayed in cerebellar cells treated with KCN to deplete ATP levels and block subsequent phosphorylation reactions of transported uridine. Uptake activity under these conditions was unaffected by MeHg. Measurement of intracellular phosphorylation of [³H]uridine indicated that inhibition of this activity closely paralleled that of RNA synthesis. Quantitation of individual uridine nucleotides by polyethyleneimine-cellulose TLC revealed reduced levels of UTP and UDP whereas levels of UMP were elevated, suggesting that impairment of phosphorylation was not the result of cellular ATP depletion but, more likely, a direct effect on phosphouridine kinase enzymes. This mechanism of MeHg-induced inhibition of RNA synthesis was confirmed by assays of uridine phosphorylation using cell-free extracts in which exogenous ATP was supplied.     

Human airway ecto-adenylate kinase. A mechanism to propagate ATP signaling on airway surfaces

Mechanically induced ATP release from human airway epithelial cells regulates mucociliary clearance through cell surface nucleotide receptors. Ectoenzymes detected on these cells were recently shown to terminate ATP-mediated responses by sequential dephosphorylation of extracellular ATP into ADP, AMP, and adenosine. We now demonstrate that an ecto-adenylate kinase (ecto-AK) contributes to the metabolism of adenine nucleotides on human airway epithelial surfaces by the reversible reaction: ATP + AMP 2ADP. This phosphotransferase exhibited a bilateral distribution on polarized primary cultures of human bronchial epithelial cells with a 4-fold higher activity on the mucosal surface. Ecto-AK presented an absolute requirement for magnesium and adenine-based nucleotides. UMP, GMP, and CMP could not substitute for AMP as gamma-phosphate acceptor, and UDP could not replace ADP. Apparent K(m) and V(max) values were 23 +/- 5 microM and 1.1 +/- 0.1 nmol x min(-1) x cm(-2) for ATP and 43 +/- 6 microM and 0.5 +/- 0.1 nmol x min(-1) x cm(-2) for ADP. Ecto-AK accounted for 20% of [gamma-(32)P]ATP dephosphorylation, and the impermeant AK inhibitor, diadenosine pentaphosphate, reduced ADPase activity by more than 70% on both epithelial surfaces. Time course experiments on ATP metabolism demonstrated that ecto-AK significantly prolongs effective ATP and ADP concentrations on airway epithelial surfaces for P2 receptor signaling and reduces by 6-fold adenosine production. Our data suggest a role for this nucleotide entrapment cycle in the propagation of purine-mediated mucociliary clearance on human airway epithelial surfaces.     

Changes in Levels of Purine and Pyrimidine Nucleotides During Acute Hypoxia and Recovery in Neonatal Rat Brain

Neonatal rat brains were examined for changes in levels of ATP, ADP, AMP, cyclic AMP, GTP, GDP, UTP, UDP, UMP, and CTP during exposure to 100% nitrogen for 20 min and subsequent recovery in air. During hypoxia, ATP, GTP, UTP, and CTP levels and the GTP/GDP ratio decreased to 38, 50, 26, 21, and 21%, respectively, of control levels. No significant change in cyclic AMP level was observed. The decrease in the total uridine nucleotide pool during hypoxia was markedly greater (to 53% of control levels) than that in the total adenine nucleotide pool (to 92% of control levels). During recovery, ATP and GTP levels were rapidly and almost completely restored. On the other hand, CTP levels returned slowly to control values after a 2-h recovery period. Restoration of the UTP level was slow and incomplete (87% of the control value even after a 3-h recovery period). The GTP/GDP ratio also did not return to normal. These data suggest that hypoxic insult to the neonate may have an effect on the synthesis of nucleotidyl sugars, phospholipids, and proteins in the brain, resulting in significant problems with developmental processes of the brain. The present study also showed that the delayed restorations of the UTP level and the GTP/GDP ratio were not seen in the brains of adult rats subjected to acute severe hypoxic insult.     

Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose

Five recombinant Escherichia coli extracts harboring overexpressed galactokinase, galactose-1-phosphate uridyltransferase, UDP-glucose pyrophophorylase, UMP kinase, and acetate kinase (AK) were utilized for the production of UDP-galactose (UDP-Gal). We analyzed the parameters which limit the yield of UDP-Gal in the reaction, and the reaction was optimized by increasing the concentration of AK. AK was used for the ATP regeneration as well as the conversion of UDP to UTP. The activities of four overexpressed enzymes were identically fixed, and then we increased the activity of AK to 20 times higher than others. The extracts catalyzed the production of UDP-Gal from UMP (10 mM), galactose (12 mM), ATP (1 mM), and acetyl phosphate (40 mM). As the result of the reaction, the conversion yield of UDP-Gal reached to 95% from 10 mM UMP.     

Glucose transport and metabolism in cultured cells of nervous tissue.

The effect of the concentration of glucose in the medium on the intracellular concentrations of metabolites of C-6 astrocytoma cells and C-1300 neuroblastoma cells in culture has been investigated. The intracellular concentrations of glucose, glycogen, glucose 6-P and UDP-glucose were measured at intervals after feeding the cells. A rapid increase in glucose and glucose 6-P levels occurred when fresh medium containing 5.5 mM glucose was applied to the cells, followed by slower increases in UDP-glucose andglycogen. When the medium glucose was increased ten-fold, the intracellular concentration of glucose was increased, but the level of glucose 6-P, UDP=-glucose and glycogen were not altered, nor were the rates of accumulation. The addition of insulin to the medium resulted in an increase of intracellular glucose, glucose 6-P and glycogen. The transport of glucose into the cells is not the rate-limiting step of the regulation of metabolite levels in the cells.     

Some Properties of Synthetic UDP(2)-fructose

UDP(2)-fructose was synthesized from d-fructofuranose-2-phosphate by the method of Khorana et al. The product thus obtained showed slightly higher paper chromatographic mobilities than those of UDP-glucose and UDP(1)-fructose, and a large negative optical rotation. In acid hydrolysis, this substance was quickly converted into UDP(1)-fructose and then the latter is hydrolyzed to UMP and fructose-1-phosphate. The rate constant of this first step is far larger than the acid hydrolysis rate constant of natural UDP-fructose isolated from the tubers of Jerusalem artichoke. When treated with the snake venom nucleotide pyrophosphatase, UDP(2)-fructose was splitted into UMP and a substance having the same paper chromatographic mobility as that of fructofuranose-2-phosphate. From these results and those reported previously, the structure of the synthetic product may be UDP(2)-β-d-fructofuranose. It was argued that the natural UDP-fructose may be UDP(2)-α-d-fructofuranose.     

Uridine Metabolism in the Goldfish Retina During Optic Nerve Regeneration: Cell-Free Preparations

The activities of uridine kinase (EC 2.7.1.48), uridine monophosphate (UMP) kinase (EC 2.7.1.3.14), and uridine diphosphate (UDP) kinase (EC 2.7.4.6) were measured in retinal high-speed supernatant fractions following unilateral optic nerve crush in the goldfish. The enzyme activities followed a similar time course, with initial increases 2-3 days following nerve crush, peak activity at 4 days, and a gradual return to basal levels by day 21. The magnitude of the stimulation on day 4 was about 35% in each case. Activities of two enzymes of intermediary metabolism, pyruvate kinase (EC 2.7.1.40) and lactic dehydrogenase (EC 1.1.1.27), were not altered, indicating that the coordinate increases in nucleoside and nucleotide kinase activities were specific responses to the nerve injury. The increased labeling could not be explained by altered phosphohydrolytic activities. The nature of the enhancement was further studied in UDP kinase, the most active of the kinases examined. Neither low-molecular-weight components nor substrate availability could account for the observed increase in UDP kinase in the 4 day post-crush retinas. The Km for UDP was unaltered, and a mixing experiment did not support the possibility that stimulatory or inhibitory factors played a role. The enhancement of UDP kinase activity was blocked by injection of actinomycin D following nerve crush. The results suggest that the observed increases in enzymes of uridine metabolism result from their increased formation following nerve crush.     

Plastid Development in Primary Leaves of Phaseolus vulgaris: FREE NUCLEOTIDE CONCENTRATIONS IN GREENING LEAVES

The free nucleotides of 14-day-old dark-grown bean leaves havebeen extracted by perchloric acid and fractionated by ion-exchangechromatography. AMP, ATP, CMP, GMP, GDP, ADP-ribosephosphate(the acid-breakdown product of NADPH), NADP⁺, UMP, UDP-glucose,and UDP-xylose were the main components identified. During 45h continuous illumination greening occured and there was a 192per cent increase in the total free nucleotide per leaf. Mostof the constituent nucleotides showed rises during greening,the most notable increases being of substances concerned withphotosynthesis, namely NADPH, and UDP-glucose. A rise in theNADPH/NADP⁺ ratio after 15 h illumination appeared to signifythe onset of photosynthesis.