Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces cerevisiae.

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effect of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is discussed.     

A mannitol teichoic acid containing rhamnose and pyruvic acid acetal from the cell wall of Brevibacterium permense VKM Ac-2280

The cell wall of Brevibacterium permense VKM Ac-2280 contains two teichoic acids. The major polymer represents a 1,6-poly(mannitol phosphate) substituted wirh either L-rhamnose (approximately 70%, unit A) or (S)-acetal of pyruvic acid (approximately 30%, unit B) with the overall chain length approximately 10 mannitol phosphate units. [carbohydrate structure: see text] The other polymer is an unsubstituted 1,3-poly(glycerol phosphate). The structures of the polymers were established using chemical degradations and NMR spectroscopy. The data obtained may be helpful in determination of the species-specific status of newly isolated Brevibacterium strains.     

Effects of pleural pressure and abdominal pressure on diaphragmatic blood flow

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.     

Structure of the cell wall proteogalactomannan from Neurospora crassa. I. Purification of the proteoheteroglycan and characterization of alkali-labile oligosaccharides

Proteoheteroglycan (PHG) was prepared from Neurospora crassa cells by extraction with hot water followed by cetyltrimethylammoniumbromide fractionation. The polymer was purified by DEAE-cellulose chromatography followed by gel filtrations. The PHG was fractionated into five subfractions containing carbohydrate (65-88%), protein (19-36%), and a trace amount of phosphate (0.3-1.9%). The sugar compositions of the fractions were similar to each other (D-mannose, 47-60%, D-galactose, 35-50%, D-glucose, 2-5%) while the fractions showed significant heterogeneity in molecular size. Mild alkali treatment of the PHG in the presence of sodium borohydride yielded three kinds of reduced oligosaccharides. Structural studies using a methylation-GC-MS method, and proton and carbon NMR indicated that the tetrasaccharide fragment is beta-D-Galf(1-5)-beta-D-Galf(1-2)-alpha-D-Manp(1-2)man nitol, the trisaccharide is beta-D-Galf(1-2)-alpha-D-Manp(1-2)mannitol, and the disaccharide is alpha-D-Manp(1-2)mannitol.     

Mannitol-specific enzyme II of the bacterial phosphotransferase system. II. Reconstitution of vectorial transphosphorylation in phospholipid vesicles

Purified mannitol Enzyme II from Escherichia coli was reconstituted in phospholipid vesicles employing the octylglucoside dilution procedure and was shown to catalyze vectorial mannitol 1-phosphate:mannitol transphosphorylation. Reconstitution of the enzyme into liposomes showed a marked dependency upon the octylglucoside concentration with an optimum at 1.2%. The reconstituted transphosphorylation activity exhibited an absolute dependence upon mannitol 1-phosphate as the phosphoryl donor, was sensitive to N-ethylmaleimide, and had a pH optimum near 6. The intravesicular radiolabeled mannitol phosphate could be released from the proteoliposomes by the addition of either 50 microM unlabeled mannitol or 0.5% sodium dodecyl sulfate. The rate of formation of intraliposomal mannitol phosphate, measured as a function of the mannitol Enzyme II concentration, showed a sigmoidal response, suggesting that at high enzyme concentrations the mannitol Enzyme II exists in an aggregated or oligomeric state and that this form is more active than the monomeric or dissociated form of the enzyme in catalyzing the vectorial mannitol transphosphorylation reaction.     

Structural studies of a mannitol teichoic acid from the cell wall of bacterium N.C.T.C. 9742.

Degradative and n.m.r.-spectroscopic studies have been carried out on a novel mannitol teichoic acid extracted from the cell wall of bacterium N.C.T.C. 9742, for which the name Brevibacterium iodinum has been proposed. The backbone of the polymer is a poly(D-mannitol phosphate) containing 1----6 phosphodiester linkages. In most residues, pyruvic acid is acetal-linked to positions 4 and 5 of the mannitol. About half of the mannitol residues carry a beta-D-glucopyranosyl substituent at position 2. The glucosylmannitol was isolated and thoroughly characterized. At least 24 products were detected by ion-exchange chromatography and paper electrophoresis after alkaline hydrolysis of the polymer. Not all of these products could be identified. The main mechanistic pathways for depolymerization by the cleavage of phosphodiester linkages during alkaline hydrolysis involved (a) participation by the 2-hydroxy group and a cyclic phosphodiester intermediate (leading to a series of mannitol-based products) and (b) participation by the 3-hydroxy group in the cyclization of mannitol (leading to a series of products based on 1,4-anhydromannitol). The presence of glycerol phosphates in hydrolysates could be ascribed either to a linkage unit or to a separate glycerol teichoic acid. The mannitol teichoic acid was absent from the cell walls of Brevibacterium linens and Brevibacterium epidermis (one strain of each was examined).     

D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions.

When Absidia glauca was grown in minimal media with D-mannitol as the only source of carbon, an NAD+ specific D-mannitol dehydrogenase (EC 1.1.1.67) was induced. The crude extract also gave evidence of mannitol kinase, mannitol-1-phosphate dehydrogenase, phosphofructokinase, and L-iditol dehydrogenase activity. The heat labile purified preparation was judged enzymically homogeneous based on evidence derived from substrate specificity studies and activity staining, following disc gel electrophoresis. The enzymic monomer, with a weight of about 67000 daltons, slowly polymerizes when stored at -20 degrees C, giving a multiplicity of protein bands on electrophoresis distributed predominantly across a spectrum from dimer to pentamer, with enzymic activity resident predominantly in even multiples of the monomer. Depolymerization occurred rapidly (hours) when a frozen preparation was brought to and held between 4 and 20 degrees C. Aggregate fragmentation with sodium dodecyl sulfate showed a time-temperature dependence, terminating in a subunit component of 13000 daltons. pH optimum for polyol oxidation occurs at 9.6 (NaOH-glycine buffer) while ketose reduction proceeded most rapidly at pH 7.0-7.2 (phosphate buffer). A regulatory role is suggested for this enzyme based on dead-end inhibition by mannitol 1-phosphate, multiple enzyme forms, and its locus at the initiation site for mannitol utilization. The physiological relevance of low-temperature aggregation to regulatory control remains to be established.     

Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces Cerevisiae

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effects of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is disscussed.     

Some effects of mannitol on the glucose metabolism of two derivatives of a strain ofRhizobium japonicum differing in mannitol dehydrogenase

The effects of mannitol were investigated by comparing some metabolic features in colonial derivatives, I-110 and L1-110, ofRhizobium japonicum strain 3IIb110, grown either on glucose alone (G-cells) or in glucose media supplemented with mannitol (GM-cells). The polyol stimulated the synthesis of not only mannitol dehydrogenase, which is active in derivative L1-110, but also the nicotinamide adenine dinucleotide (NAD)-linked 6-phosphogluconate (6-PG) dehydrogenase (EC 1.1.1.43). As revealed by radiorespirometry, when GM-cells were allowed to metabolize glucose, they produced relatively more CO₂ from the first and sixth carbons of the sugar than G-cells did. This finding is evidence that NAD-linked 6-PG dehydrogenase might initiate an unknown pathway different from the hexose cycle and the pentose phosphate (PP) pathway. Mannitol exerted no allosteric control on the oxygen consumption and the glucose transport systems. Active uptake of the polyol was correlated with the presence of mannitol dehydrogenase (EC 1.1.1.67); it did not hinder the transport of glucose even though both systems derive their energy for active transport from a common source presumptively characterized as the energized membrane state. Mannitol, however, suppressed by two- or threefold the glucose uptake system. Addition of the polyol to the cell suspensions of both colonial types ofR. japonicum metabolizing glucose caused an immediate 40–50% drop of adenosine triphosphate (ATP) concentrations, owing in part to the mannitol kinase reaction. Type I-110 failed to overcome this reduction of ATP levels, and low growth rates could results. In contrast, type L1-110 offsets the reduction of ATP concentration by oxidizing mannitol as an additional source of energy through mannitol dehydrogenase, fructokinase, and a sequence of glycolytic reactions. The polyol also induced type L1-110 to produce extracellular slimy materials that, apparently, harbor amounts of ATP and proteins.     

Structures of cell wall teichoic acids of <Emphasis Type="Italic">Brevibacterium iodinum</Emphasis> VKM Ac-2106<Superscript>T</Superscript>

Structures of two cell wall teichoic acids of Brevibacterium iodinum VKM Ac-2106^T were studied. The structure of mannitol teichoic acid described earlier was mainly confirmed. This polymer is 1,6-poly(mannitol phosphate) bearing -D-glucopyranosyl residues at the C-2 of mannitol and pyruvic acid residues at the C-4 and C-5. The absolute configurations of D-mannitol and S-pyruvic acid were found. The following distinctions from the earlier described structure were found: unsubstituted 1,6-poly(mannitol phosphate) residues and residues substituted only by -D-glucopyranosyl at the C-2 of mannitol but unsubstituted by pyruvic acid are present in the chain. The structure of glycerol teichoic acid present in the cell wall as a minor component (7%) is also described. This acid is identified as 1,3-poly(glycerol phosphate) substituted at the C-2 of glycerol by 2-acetamido-2-deoxy--D-galactopyranosyl residues bearing R-pyruvic acid residues at the C-4 and C-6 of galactose. This polymer is for the first time described in the cell wall of Gram-positive bacteria.Translated from Biokhimiya, Vol. 69, No. 12, 2004, pp. 1659–1666.Original Russian Text Copyright © 2004 by Potekhina, Evtushenko, Senchenkova, Shashkov, Naumova.     

Rapid turnover of mannitol-1-phosphate in Escherichia coli.

The phosphate moiety of D-mannitol-1-phosphate in Escherichia coli is subject to rapid turnover and is in close equilibrium with Pi and the phosphorus of fructose-1,6-bisphosphate. These three compounds account for the bulk of 32P label found in cells after several minutes of uptake of 32Pi and mannitol-1-phosphate represents some 30% of this label. Mannitol-1-phosphate occurs in E. coli grown on a variety of carbon sources, in the absence of D-mannitol, and is synthesized de novo even in mutants lacking mannitol-1-phosphate dehydrogenase. The mannitol moiety of mannitol-1-phosphate was not affected during the total chase of the P moiety, which exchanged with a half-life of about 30 s. These findings suggest that the rapid equilibration of the phosphorus is a function of an enzyme, possibly a component of the phosphotransferase system, capable of forming a complex that allows the exchange of the phosphate without the equilibration of the mannitol moiety with free mannitol.     

Crystal and molecular structure of sym-homospermidine monohydrate.

Sym-homospermidine, [formula; see text] is a naturally occurring rare-polyamine found in relatively large concentration in sandal leaves. As part of our studies on structure and interactions of polyamines, sym-homospermidine was purified from sandal leaves and its structure was determined by single crystal X-ray diffraction technique. The phosphate salt of the molecule crystallized in the triclinic space group P1- with a = 8.246(1)A, b = 8.775(1)A, c = 15.531(2)A, alpha = 74.20(1) degrees, beta = 88.36(1) degrees and gamma = 65.41(1) degrees. The structure was determined by direct methods and refined to a final R factor of 5.4% for 2087 reflections with magnitude of F(obs) greater than 5 sigma [F(obs)]. The amine exists in its most favourable all trans conformation. For each amine molecule three phosphate groups exist in the crystal structure, suggesting that two of the oxygens of each phosphate group are protonated. There is also a single water molecule in the asymmetric unit in contrast to that of spermidine phosphate which has 3 water molecules. These differences probably reflect the hydrogen bonding properties of mono-ionic and di-ionic phosphate groups. The structure is predominantly stabilized by a network of hydrogen bonds.     

Mannitol‐1‐phosphate dehydrogenases/phosphatases: a family of novel bifunctional enzymes for bacterial adaptation to osmotic stress

The nutritionally versatile soil bacterium Acinetobacter baylyi ADP1 copes with salt stress by the accumulation of compatible solutes, a strategy that is widespread in nature. This bacterium synthesizes the sugar alcohol mannitol de novo in response to osmotic stress. In a previous study, we identified MtlD, a mannitol‐1‐phosphate dehydrogenase, which is essential for mannitol biosynthesis and which catalyses the first step in mannitol biosynthesis, the reduction of fructose‐6‐phosphate (F‐6‐P) to the intermediate mannitol‐1‐phosphate (Mtl‐1‐P). Until now, the identity of the second enzyme, the phosphatase that catalyses the dephosphorylation of Mtl‐1‐P to mannitol, was elusive. Here we show that MtlD has a unique sequence among known mannitol‐1‐phosphate dehydrogenases with a haloacid dehalogenase (HAD)‐like phosphatase domain at the N‐terminus. This domain is indeed shown to have a phosphatase activity. Phosphatase activity is strictly Mg²⁺ dependent. Nuclear magnetic resonance analysis revealed that purified MtlD catalyses not only reduction of F‐6‐P but also dephosphorylation of Mtl‐1‐P. MtlD of A. baylyi is the first bifunctional enzyme of mannitol biosynthesis that combines Mtl‐1‐P dehydrogenase and phosphatase activities in a single polypeptide chain. Bioinformatic analysis revealed that the bifunctional enzyme is widespread among Acinetobacter strains but only rarely present in other phylogenetic tribes.     

The limitation of glycolysis in adenine-deficient Escherichia coli

1. In many instances, there was no change in the rate of oxygen consumption per cell when adenine was withdrawn from purine auxotrophs of Escherichia coli and Salmonella typhimurium. 2. However, adenine deficiency inhibited the metabolism of glucose, mannitol or glycerol in a purA(-) strain, in purB(-) or purH(-) strains in the absence of histidine and in purB(-) mutants supplied with hypoxanthine. These are all instances where reactions occur to consume adenine nucleotides. 3. The inhibition of glucose oxidation is accompanied by the accumulation of fructose 1,6-diphosphate and dihydroxyacetone phosphate. 4. Insufficiency of ADP for phosphoglycerate kinase is the most probable cause of the inhibition.     

The structure of a mannitol teichoic acid from Bifidobacterium bifidum ssp. Pennsylvanicum

The isolation and analysis of the cell wall and cell wall fractions of Bifidobacterium bifidum ssp. pennsylvanicum are presented. With lysozyme a solubilized cell wall fraction is obtained which contains muramic acid, glucosamine, rhamnose, glucose, mannitol, phosphate and all peptidoglycan amino acids. Its composition did not change with culture age. A glycogen-like glucose polymer which is of cytoplasmic origin is identified in the insoluble cell wall fraction. The solubilized cell wall fraction contains a glucosylated rhamnose polymer which is linked by glycosidic bonds to the peptidoglycan fragments. This polymer is a 1,2-linked or an alternating, 1,2/1,3-linked α-rhamnose chain substituted on average at every second rhamnose residue with an α-linked glucose molecule. Various experiments gave evidence that mannitol and phosphate are present in 4,6-linked mannitol phosphate oligomers which are linked by phosphodiester bonds to the glucosylated rhamnose polymer. These oligomers may fulfill the functions of the more common wall teichoic acids.     

Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions

Column chromatography of the Escherichia coli mannitol permease (mannitol-specific enzyme II of the phosphotransferase system) in the presence of deoxycholate has revealed that the active permease can exist in at least two association states with apparent molecular weights consistent with a monomer and a dimer. The monomeric conformation is favored by the presence of mannitol and by the phosphoenolpyruvate (PEP)-dependent phosphorylation of the protein. The dimer is stabilized by inorganic phosphate (Pi), which also stimulates phospho-exchange between mannitol and mannitol 1-phosphate (a partial reaction in the overall PEP-dependent phosphorylation of mannitol). Kinetic analysis of the phospho-exchange reaction revealed that Pi stimulates phospho-exchange by increasing the Vmax of the reaction. A kinetic model for mannitol permease function is presented involving both conformations of the permease. The monomer (or a less-stable conformation of the dimer) is hypothesized to be involved in the initial mannitol-binding and PEP-dependent phosphorylation steps, while the stably associated dimer is suggested to participate in later steps involving direct phosphotransfer between the permease, mannitol and mannitol 1-phosphate.     

Effect of parathyrin on the transport properties of isolated renal brush-border vesicles.

The transport properties of brush-border membrane vesicles isolated by a calcium-precipitation method from the renal cortex of normal and parathyrin (parathyroid hormone)-treated rats were studied by a rapid-filtration technique. Parathyrin elicited a dose-dependent decrease in the Na+-dependent phosphate uptake by the brush-border membrane vesicles, but the uptake of D-glucose, Na+ and mannitol was not affected. A maximum inhibition of 30% was observed after the application of 30 U.S.P. units intramuscularly 1 h before the animals were killed. Intravenous infusion of dibutyryl cyclic AMP (0.5-1.5 MG) also decreased the phosphate uptake by the brush-border vesicles. Both dibutyryl cyclic AMP and parathyrin were ineffective when added in vitro to brush-border membrane vesicles isolated from normal rats. These data suggest that parathyrin exerts its action on the phosphate reabsorption in the renal proximal tubule by affecting the Na+/phosphate co-transport system in the brush-border membrane. The effects of parathyrin on Na+ and glucose transport, however, seem to be due to alterations to the driving forces for transport and not to the brush-border transport systems.     

Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties.

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.     

Characterization of the Streptococcus adjacens group antigen structure.

Serological classification of bacteria requires the presence of an antigen unique to the organism of interest. Streptococci are serologically differentiated by group antigens, many of which are carbohydrates, although some are amphiphiles. This report describes the chemical characterization of the Streptococcus adjacens group antigen structure. Previous studies demonstrated that the amphiphile contained phosphorus, ribitol, galactose, galactosamine, alanine, and fatty acids. Phosphodiester bonds present in the purified group antigen were identified as part of a poly(ribitol phosphate), since ribitol phosphate was the only organic phosphate detected after acid hydrolysis. Hydrofluoric acid cleavage of the phosphodiester bonds generated oligosaccharide repeating units. Gas chromatography-mass spectrometric analysis of the methylated, acetylated oligosaccharide suggested that the repeating unit is a trisaccharide of Galp beta 1-3Galp beta 1-4GalNac with N-acetylgalactosamine attached in beta-linkage to either the number two or the number four carbon of ribitol. The lipid- and carbohydrate-substituted poly(ribitol phosphate) of the S. adjacens group antigen therefore is a unique amphiphile structure, differing in its repeating-unit structure from the polyglycerophosphate structure of the more common gram-positive amphiphile lipoteichoic acid.