(1/53) Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.
Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels. (+info)
(2/53) Composition and biological properties of lipopolysaccharides isolated from Schizothrix calcicola (Ag.) Gomont (Cyanobacteria).
The most common cyanobacterium contaminating drinking water systems in southwestern Pennsylvania is Schizothrix calcicola. Lipoplysaccharides (LPS) were isolated from this species by hot phenol-water extraction. The polysaccharide moiety was composed of glucosamine, galactose, glucose, mannose, xylose and rhamnose. The lipid A part contained beta-hydroxylauric, myristic, pentadecanoic, palmitic, beta-hydroxypalmitic, stearic, oleic, and linoleic acids. In contrast to many LPS isolated from Enterobacteriaceae, the dominant component was not beta-hydroxymyristic but beta-hydroxypalmitic acid. The LPS induced Limulus lysate gelation and Schwartzman reaction but was nontoxic to mice. The identity of LPS was verified by alkali and lysozyme treatment. The results suggest that S. calcicola is one of the principal sources of endotoxins in water systems using open finished-water reservoirs. (+info)
(3/53) 3,4-Dideoxyglucosone-3-ene (3,4-DGE): a cytotoxic glucose degradation product in fluids for peritoneal dialysis.
BACKGROUND: Bioincompatible glucose degradation products (GDPs) in fluids for peritoneal dialysis (PD) develop during sterilization and storage. Their biological activity has successfully been monitored through the use of various in vitro methods but their molecular and chemical nature is less well understood. Many GDPs are highly reactive carbonyl compounds. Although some of the identified GDPs are extremely cytotoxic, none of them actually possess cytotoxicity at the concentrations found in PD fluids. Thus, the GDP responsible for the toxicity in PD fluids has not yet been identified. The intention of the present work was to investigate to what extent the unsaturated dicarbonyl compound, 3,4-dideoxyglucosone-3-ene (3,4-DGE) was present in PD fluids, and if it could be responsible for the in vitro effects on L-929 fibroblast cells. METHODS: A commercial preparation of 3,4-DGE and two different liquid chromatography methods were used for the chemical identification and quantification. In vitro bioincompatibility was determined as inhibition of cell growth using the L-929 fibroblast cell line. RESULTS: 3,4-DGE was present in conventionally manufactured PD fluids at a concentration of 9 to 22 micromol/L. In the newly developed PD fluid, Gambrosol trio, the concentrations were 0.3 to 0.7 micromol/L. When added as synthetic 3,4-DGE to cell growth media at the concentrations measured in conventional PD fluids, the inhibition of cell growth was significantly lower than for that seen with the conventional fluids. However, in the conventional PD fluids the total amount of 3,4-DGE available for toxic reactions most probably was higher than that measured, because 3,4-DGE was freshly recruited from a molecular pool when consumed. The speed of this recruitment was high enough to explain most of the growth inhibition seen for heat-sterilized PD fluids. CONCLUSION: 3,4-DGE is present in conventional PD fluids at a concentration between 9 and 22 micromol/L, and is the most biologically active of all GDPs identified to date. Thus, it is the main candidate to be held responsible for the clinical bioincompatibility caused by conventionally manufactured PD fluids. (+info)
(4/53) Peroxynitrite induces formation of N( epsilon )-(carboxymethyl) lysine by the cleavage of Amadori product and generation of glucosone and glyoxal from glucose: novel pathways for protein modification by peroxynitrite.
Accumulation of advanced glycation end products (AGEs) on tissue proteins increases with pathogenesis of diabetic complications and atherosclerosis. Here we examined the effect of peroxynitrite (ONOO(-)) on the formation of N( epsilon )-(carboxymethyl)lysine (CML), a major AGE-structure. When glycated human serum albumin (HSA; Amadori-modified protein) was incubated with ONOO(-), CML formation was detected by both enzyme-linked immunosorbent assay and high-performance liquid chromatography (HPLC) and increased with increasing ONOO(-) concentrations. CML was also formed when glucose, preincubated with ONOO(-), was incubated with HSA but was completely inhibited by aminoguanidine, a trapping reagent for alpha-oxoaldehydes. For identifying the aldehydes that contributed to ONOO(-)-induced CML formation, glucose was incubated with ONOO(-) in the presence of 2,3-diaminonaphthalene. This experiment led to identification of glucosone and glyoxal by HPLC. Our results provide the first evidence that ONOO(-) can induce protein modification by oxidative cleavage of the Amadori product and also by generation of reactive alpha-oxoaldehydes from glucose. (+info)
(5/53) Isolation and characterization of membranes from a hydrocarbon-oxidizing Acinetobacter sp.
Membranes were isolated and purified from nutrient broth-yeast extract- and hexadecane-grown cells of Acinetobacter sp. strain HO1-N. Two membrane fractions were isolated from nutrient broth-yeast extract-grown cells, the cytoplasmic membrane and the outer membrane. In addition to these two membrane fractions, a unique membrane fraction was isolated from hexadecane-grown cells (band 1) and characterized as a lipid-rich, low-density membrane containing high concentrations of hexadecane. The outer membrane preparations of Acinetobacter, obtained from nutrient broth-yeast extract- and hexadecane-grown cells, exhibited a low ratio of lipid phosphorus to protein and contained phospholipase activity and 2-keto-3-deoxyoctulosonic acid. Phosphatidic acid cytidyltransferase, adenosine triphosphatase, and reduced nicotinamide adenine dinucleotide oxidase were recovered almost exclusively in the cytoplasmic membrane fractions. The cytoplasmic membrane fractions contained 20 to 25 polypeptide species on sodium dodecyl sulfate-polyacrylamide gels, and the outer membrane fractions contained 15 to 20 polypeptide species. A major polypeptide species with an apparent molecular weight of approximately 42,000 to 44,000 was found for all outer membrane fractions. The buoyant densities of the cytoplasmic membrane fractions and the outer membrane fractions were closely similar, necessitating their separation by differential centrifugation. Band 1 of hexadecane-grown cells had a ratio of lipid phosphorus to protein that was almost twice that of cytoplasmic membrane and a correspondingly low buoyant density (1.086 g/cm3). Enzyme activities associated with band 1 were identical to those associated with the cytoplasmic membrane. The electrophoretic banding pattern of band 1 was essentially identical to the banding pattern of the cytoplasmic membrane. The phospholipid and neutral lipid compositions of the isolated membrane fractions were determined as qualitatively similar, with significant quantitative differences. The ultrastructure characteristics of the respective membrane fractions were examined by the negative-stain technique. (+info)
(6/53) Purification, characterization, and molecular cloning of a pyranose oxidase from the fruit body of the basidiomycete, Tricholoma matsutake.
A new H(2)O(2)-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H(2)O(2) and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V(max), K(m), and k(cat) for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50 degrees C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody. (+info)
(7/53) The large subunit determines catalytic specificity of barley sucrose:fructan 6-fructosyltransferase and fescue sucrose:sucrose 1-fructosyltransferase.
Plant fructosyltransferases are highly homologous in primary sequence and typically consist of two subunits but catalyze widely different reactions. Using functional expression in the yeast Pichia pastoris, we show that the substrate specificity of festuca sucrose:sucrose 1--beta-D-fructosyltransferase (1-SST) and barley sucrose:fructan 6--beta-D-fructosyltransferase (6-SFT) is entirely determined by the large subunit. Chimeric enzymes with the large subunit of festuca 1-SST (LSuB) and the small subunit of barley 6-SFT have the same catalytic specificity as the native festuca 1-SST and vice versa. If the LSuB is expressed alone, it does not yield a functionally active enzyme, indicating that the small subunit is nevertheless essential. (+info)
(8/53) Enzymatic description of the anhydrofructose pathway of glycogen degradation II. Gene identification and characterization of the reactions catalyzed by aldos-2-ulose dehydratase that converts 1,5-anhydro-D-fructose to microthecin with ascopyrone M as the intermediate.
The anhydrofructose pathway describes the degradation of glycogen and starch to metabolites via 1,5-anhydro-D-fructose (1,5AnFru). Enzymes that form 1,5AnFru, ascopyrone P (APP), and ascopyrone M (APM) have been reported from our laboratory earlier. In the present study, APM formed from 1,5AnFru was found to be the intermediate to the antimicrobial microthecin. The microthecin forming enzyme from the fungus Phanerochaete chrysosporium proved to be aldos-2-ulose dehydratase (AUDH, EC 4.2.1.-), which was purified and characterized for its enzymatic and catalytic properties. The purified AUDH showing a molecular mass of 97.4 kDa on SDS-PAGE was partially sequenced. Total 332 amino acid residues in length were obtained, representing some 37% of the AUDH protein. The obtained amino acid sequences showed no homology to known proteins but to an unannotated DNA sequence in Scaffold 62 of the published genome of the fungus. The alignment revealed three introns of the identified AUDH gene (Audh; ph.chr), thus the first gene coding for a neutral sugar dehydratase is identified. AUDH was found to be a bi-functional enzyme, being able to dehydrate 1,5AnFru to APM and further isomerizing the APM formed to microthecin. The optimal pH for the formation of APM and microthecin was pH 5.8 and 6.8, respectively. AUDH showed 5 fold higher activity toward 1,5AnFru than toward its analogue glucosone, when tested at concentrations from 0.6 mM to 0.2 M. Based on the characteristic UV absorbance of microthecin (230 nm) and APM (262 nm) assay methods were developed for the microthecin forming enzymes. (+info)