Protein histidine phosphorylation: increased stability of thiophosphohistidine.
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Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins has been discovered in prokaryotic organisms as well as in eukaryotic cells. The ability to biochemically analyze phosphohistidine residues in proteins, however, is severely hampered by its extreme lability under acidic conditions. In our studies we have found that by replacing the phosphate linked to the histidine residue with a thiophosphate, a phosphohistidine derivative with increased stability is formed. This allows the analysis of phosphohistidine-containing proteins by established biochemical techniques and will greatly aid in the investigation of the role of this posttranslational modification in cellular processes. (+info)
Nucleotide sequence of an RNA polymerase binding site from the DNA of bacteriophage fd.
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The primary structure of a strong RNA polymerase binding site in the replicative form DNA of phage fd has been determined by direct DNA sequencing. It is: (see article). The molecule contains regions with 2-fold symmetry and sequence homologies to promoter regions from other DNAs. The startpoint of transcription is located in the center of the binding site. (+info)
Re-evaluation of plant sulpholipid labelling from UDP-[14C]glucose in pea chloroplasts.
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The sulphoquinovosyldiacylglycerol (sulpholipid) zone obtained by TLC of pea chloroplast lipids yields, on hydrolysis, not only sulphoquinovose but also galactose and glucose. Following incorporation from UDP-[(14)C]glucose, the percentages of the total radioactivity in these three sugars were typically 1, 5 and 85%, respectively. The occurrence of the glucolipid causes difficulties in elucidating further the biosynthetic pathway for sulphoquinovose. (+info)
Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment.
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Assessment of free fatty acid (FFA) concentration and isotopic enrichment is useful for studies of FFA kinetics in vivo. A new procedure to recover the major FFA from plasma for concentration and isotopic enrichment measurements is described and validated. The procedure involves extraction of plasma lipids with hexane, methylation with iodomethane (CH(3)I) to form fatty acid methyl esters (FAME), and subsequent purification of FAME by solid phase extraction (SPE) chromatography. The new method was compared with a traditional method using thin-layer chromatography (TLC) to recover plasma FFA, with subsequent methylation by BF(3)/methanol. The TLC method was found to be less reliable than the new CH(3)I method because of contamination with extraneous fatty acids, chemical fractionation of FFA species, and incomplete recovery of FFA associated with TLC. In contrast, the CH(3)I/SPE method was free of contamination, did not exhibit chemical fractionation, and had higher recovery. The iodomethane reaction was specific for free fatty acids; no FAME were formed when esterified fatty acids (triglycerides, cholesteryl esters, phospholipids) were subjected to the methylation reaction. We conclude that the CH(3)I/SPE method provides rapid and convenient recovery of plasma fatty acids for quantification or GC/MS analysis as methyl esters, and is not subject to the problems of contamination, reduced recovery, and chemical fractionation associated with recovery of FFA by TLC. (+info)
Purine metabolism in murine virus-induced erythroleukemic cells during differentiation in vitro.
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Purine metabolism was studied in murine virus-induced erythroleukemia cells stimulated to differentiate in vitro in the presence of dimethylsulfoxide. The activities of the enzymes that catalyze the synthesis of the first intermediate of the de novo purine pathway, phosphoribosyl-1-amine, were decreased while the enzymes that catalyze the conversion of purine bases to purine ribonucleotides remained unchanged at the time the cells acquired the specialized function of hemoglobin synthesis. In addition, cytidine deaminase (cytidine aminohydrolase, EC 3.5.4.5) activity increased with erythropoietic maturation, as it does during murine erythropoiesis in vivo. Stimulation of cellular proliferation of stationary erythroleukemic cells resulted in a marked increase in the activities of purine biosynthetic enzymes. These data provide a convincing example of repression and derepression of the PRA synthesizing enzymes in mammalian cells in vitro, and further evidence that the regulatory mechanisms operative in the normal development of erythrocytes can be activated by exposure of erythroleukemic cells to dimethylsulfoxide. (+info)
Sympathetic potentiation of cyclic ADP-ribose formation in rat cardiac myocytes.
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We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation. (+info)
Neu5Acalpha3Gal is part of the Helicobacter pylori binding epitope in polyglycosylceramides of human erythrocytes.
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The sialic acid dependent binding by the human pathogen Helicobacter pylori to polyglycosylceramides of human erythrocytes was investigated. Polyglycosylceramides, complex glycosphingolipids with a branched N-acetyllactosamine core, were isolated from human erythrocytes, blood group O, and subfractionated after peracetylation by anion-exchange chromatography. Three subfractions were deacetylated, analysed by matrix-assisted laser desorption ionization-time of flight MS and 2D 1H NMR spectroscopy. The observed mass ranges were m/z = 3093-7622, 3968-7255 and 3459-7987 in the mass spectra of the first, second and third fractions, respectively. The observed ions agreed with the general formula Hex(x+2)HexNAcxFucyNeu5AczCer. Two-dimensional 1H total correlation spectra of the mixtures showed that the first fraction contained 3-linked sialic acid and the second and third fractions contained both 3-linked and 6-linked sialic acid. Thin-layer chromatogram binding assays using the lectins from Maackia amurensis, specific for Neu5Acalpha3Galbeta4GlcNAc, and Sambucus nigra, specific for Neu5Acalpha6Gal/GalNAc, were used to confirm this distribution. H. pylori recognized all three fractions in the binding assay, indicating that the 3-linked, rather than 6-linked, sialic acid is essential for binding. (+info)
Clostridium innocuum: a glucoseureide-splitting inhabitant of the human intestinal tract.
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Glycosylureides were recently described as non-invasive markers of intestinal transit time. The underlying principle is an enzymatic splitting of (13)C-labelled ureides by intestinal bacteria. The (13)CO(2) released from the urea moiety of the glycosylureides can be measured in breath samples when the ingested tracer substrate reaches the caecum that is colonised with microbes. To date, the microbes that degrade glycosylureides are unknown. In order to identify the glucoseureide (GU)-splitting bacteria, 174 different strains of intestinal microbes obtained from five healthy adults were checked for their ability to degrade GU. The results of the microbial cultures and thin layer chromatography revealed that GU was exclusively degraded by Clostridium innocuum, belonging to the normal human intestinal microflora. C. innocuum probably synthesises a yet unknown enzyme that splits the glucose-urea bond. We suggest that the term glucoseureidehydrolase is the appropriate designation for this enzyme. (+info)