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At least 127 records · Page 7Linked to original sources

PubMed1980

[Streptomycin-3"-phosphotransferases from streptomycin-resistant cells of Escherichia coli strains].

Streptomycin-3"-phosphotransferases were isolated and purified from E. coli cells containing plasmids 836, pBS52 or R6K, which determine the microorganisms resistance towards streptomycin and dihydrostreptomycin. Phosphorylation of the 3"-hydroxylic group of dihydrostreptomycin was demonstrated by [13C]-NMR spectrometry. It was shown that streptomycin-3"-phosphotransferase, whose synthesis is determined by plasmid 836 (as well as by plasmid R6K), differs from the analogous enzyme, whose synthesis is operated by plasmid pBS52 in some properties, e. g. dependence of the initial reaction rate on concentrations of antibiotics and ATP, pH-optimum, sensitivity to the buffer ionic strength, stability, etc. Besides, the antiserum against streptomycin-3'-phosphotransferase detected by plasmid pBS52 does not produce cross immunological reactions with the other enzyme.

Dihydrostreptomycin Sulfate↗

PubMed1975

The bacterial phosphoenolpyruvate: sugar phosphotransferase system.

The bacterial phosphotransferase system participates in diverse physiological phenomena; its best characterized function is in the group translocation of sugars that are substrates of the system. Such sugars are phosphorylated as they are translocated across the cell membrane. Isolation of different proteins of the phosphotransferase system and reconstitution of the complex shows that in the net transfer of the phosphoryl group from phosphoenolpyruvate to a given sugar the phosphoryl group is sequentially transferred from one protein to another. In all cases so far studied, with one important exception, the phosphoryl group is linked to the proteins through a nitrogen atom in the imidazole ring of a histidyl residue. In the exceptional protein, the phosphoryl group is linked to a carboxy group. An additional function of the phosphotransferase system is to regulate the uptake of sugars that cannot be phosphorylated.

Bacteria↗

PubMed1982

Regulation of lactose transport by the phosphoenolpyruvate-sugar phosphotransferase system in membrane vesicles of Escherichia coli.

Regulation of lactose uptake by the phosphoenolpyruvate-sugar phosphotransferase system (PTS) has been demonstrated in membrane vesicles of Escherichia coli strain ML 308-225. Substrates of the phosphotransferase system inhibited D-lactate energized uptake of lactose but did not inhibit uptake of either L-alanine or L-proline. This inhibition was reversed by intravesicular (but not extravesicular) phosphoenolpyruvate. Lactose uptake was also inhibited by enzyme IIIglc preparations that were shocked into the vesicles, and this inhibition was reversed by phosphoenolpyruvate. Intravesicular HPr and enzyme I stimulated methyl alpha-glycoside uptake but did not inhibit or stimulate lactose accumulation. Vesicles maintained at 0 degree C for several days partially lost 1) the ability to take up lactose, 2) the ability to accumulate PTS substrates, and 3) PTS-mediated regulation. Phosphoenolpyruvate addition restored all of these activities. These results support a mechanism in which the relative proportions of phosphorylated and nonphosphorylated forms of a phosphotransferase constituent regulate the activity of the lactose permease.

Bacterial Proteins↗

PubMed1997

The murine cytomegalovirus (MCMV) homolog of the HCMV phosphotransferase (UL97(pk)) gene.

The murine cytomegalovirus (MCMV) M97 gene is homologous with both eukaryotic protein kinases and the phosphotransferases of herpesviruses. The gene conserves the domain structure of protein kinases and of the human cytomegalovirus UL97 (phosphotransferase) gene. An M97 transcript of 2.5 kb is present predominantly at late times, and much smaller quantities of the transcript are detected at early times postinfection. Comparison of the DNA sequences of the complete M97 genes from 12 ganciclovir-sensitive and aciclovir-sensitive strains of MCMV showed that the sensitive isolates strongly conserve the sequence of the catalytic domains, but have only moderate conservation of the sequence of the amino-terminal (regulatory) region. MCMV provides a useful model for studying the in vivo function of the phosphotransferase genes of the betatherpesviruses and has potential for use in studies of antiviral resistance.

Amino Acid Sequence↗

PubMed1978

Studies of rat brain choline ethanolamine phosphotransferases using labeled alkylacylglycerol as substrate with evidence for reversibility of the reactions.

Cholinephosphotransferase activity in brain microsomes may be assayed with labeled alkylacylglycerols or with CDP-choline with label in the phosphocholine with nearly identical results. The direct linear plot method was used for evaluation of Michaelis-Menten kinetic parameters. Most of the cholinephosphotransferase activity is in microsomes and a stimulatory factor seems to be present in the cytosol. Incubation of microsomes with labeled alkylacylglycerols and CDP-choline, in the initial absence of CDP-ethanolamine, produced labeled ethanolamine glycerophospholipids as well as labeled choline glycerophospholipids. Since the labeling of ethanolamine glycerophospholipids was increased by the addition of CMP, the labeling was probably due to the reversal of ethanolamine phosphotransferase to yield CDP-ethanolamine produced by the choline phosphotransferase reaction. Cholinephosphotransferase was reversed more readily than ethanolaminephosphotransferase in brain as it is in liver (Kanok and Ohno, 1973). Only trace quantities of plasmalogens were formed with labeled alkylacylglycerols. Previous results of plasmalogen labeling from labeled CDP-nucleotides were apparently due to reversal of phosphotransferase reactions. Alkylacylglycerophospholipids are not good substrates for plasmalogen formation, even when they are incorporated into microsomes.

Animals↗

PubMed1983

Expression of a neomycin phosphotransferase gene from Streptomyces fradiae in Escherichia coli after interplasmidic recombination.

Plasmid pIJ2 carrying the neomycin phosphotransferase gene of Streptomyces fradiae was fused to E. coli plasmid pBR325 and the hybrid molecules were introduced into E. coli K12 by transformation. The neomycin phosphotransferase gene of the hybrid plasmid was not expressed in E. coli, except after interplasmidic recombination. Physical analysis of such an in vivo recombinant plasmid revealed that the recombination brought one neomycin phosphotransferase gene to a position downstream from the tet-promoter of pBR325. Subcloning experiments indicated that this is the gene copy expressed, and that transcription is initiated at the tet-promoter of pBR325.

Escherichia coli↗

PubMed1984

The effect of embryological development on phosphatidylethanolamine methyltransferase, phosphatidyldimethylethanolamine methyltransferase and choline phosphotransferase of rabbit liver microsomes.

The effect of embryological development on the two biosynthetic enzymes involved in phosphatidylcholine biosynthesis in liver microsomes of -12, -9, 0, +4, +14, +36 day old rabbits has been determined. The specific activity (pmol phosphatidylcholine formed/min/mg microsomal protein) of the phosphatidylethanolamine methyltransferase in the liver microsomes is very low before birth and a 33% increase at birth occurs when compared to the -12 day old fetal livers. The pmol of phosphatidylcholine formed/min/mg protein by the choline phosphotransferase pathway in fetal liver microsomes is 5, 10, 73, 199, 107 and 307 times greater than by the phosphatidylethanolamine methyltransferase pathway for -12, -9, 0, +4, +14, +36 day old rabbits, respectively. The specific activities of the choline phosphotransferase in the liver microsomes increased from the -12 day old fetal livers to 1.6, 19, 73, 39, 27 times for the -9, 0, +4, +14 and +36 day old animals, respectively. The choline phosphotransferase pathway in comparison to the phosphatidylethanolamine methyltransferase pathway is providing the major phosphatidylcholines in the membranes of the endoplasmic reticulum before birth and early fetal development of the rabbit.

Animals↗

PubMed1993

Membrane-bound 5'-nucleotidase/nucleoside phosphotransferase from Bacillus cereus.

1. A search for nucleoside phosphotransferase activity in Bacillus cereus led to the following results: (i) The phosphotransferase activity was associated with a membrane bound 5'-nucleotidase. (ii) The enzyme phosphorylates both purine and pyrimidine nucleosides as well as 2',3'-dideoxyinosine. (iii) The enzyme was inhibited by adenylic nucleotide di- and triphosphates, and its nucleotidase activity was increased in the presence of inosine as phosphate acceptor. 2. Bacterial and vertebrate 5'-nucleotidases with phosphotransferase activity differ for several characteristics, such as cellular location, substrate specificity, magnesium requirement and regulation.

5'-Nucleotidase↗

PubMed1990

Levanase operon of Bacillus subtilis includes a fructose-specific phosphotransferase system regulating the expression of the operon.

The levanase gene (sacC) of Bacillus subtilis is the distal gene of a fructose-inducible operon containing five genes. The complete nucleotide sequence of this operon was determined. The first four genes levD, levE, levF and levG encode polypeptides that are similar to proteins of the mannose phosphotransferase system of Escherichia coli. The levD and levE gene products are homologous to the N and C-terminal part of the enzyme IIIMan, respectively, whereas the levF and levG gene products have similarities with the enzymes IIMan. Surprisingly, the polypeptides encoded by the levD, levE, levF and levG genes are not involved in mannose uptake, but form a fructose phosphotransferase system in B. subtilis. This transport is dependent on the enzyme I of the phosphotransferase system (PTS) and is abolished by deletion of levF or levG and by mutations in either levD or levE. Four regulatory mutations (sacL) leading to constitutive expression of the lavanase operon were mapped using recombination experiments. Three of them were characterized at the molecular level and were located within levD and levE. The levD and levE gene products that form part of a fructose uptake PTS act as negative regulators of the operon. These two gene products may be involved in a PTS-mediated phosphorylation of a regulator, as in the bgl operon of E. coli.

Amino Acid Sequence↗

PubMed1978

Insulin action on Escherichia coli. Regulation of the adenylate cyclase and phosphotransferase enzymes.

Insulin on Escherichia coli was studied using wild type E. coli B/r and K12 strains and a number of phosphoenolpyruvate phosphotransferase mutants. In vivo, the effects of insulin on the differential rate of tryptophanase synthesis, the rate of alpha-methylglucoside uptake and the rate of growth on glucose were determined in E. coli B/r. In vitro, the effect of insulin on the adenylate cyclase and the phosphotransferase activities was determined using toluenized cell preparations of E. coli B/r, E. coli K12 and phosphotransferase mutant strains. The specificity of insulin action on E. coli was determined using glucagon, vasopressin and somatropin as well as insulin antisera. Results show the specific action of insulin on E. coli, inhibiting tryptophanase induction and adenylate cyclase activity, while stimulating growth on glucose and uptake and phosphorylation of alpha-methylglucoside.

Adenylyl Cyclases↗

PubMed2004

Helical shifts generate two distinct conformers in the atomic resolution structure of the CheA phosphotransferase domain from Thermotoga maritima.

Helical histidine phosphotransferase (HPt) domains play a central role in many aspects of bacterial signal transduction. The 0.98 A resolution crystallographic structure of the amino-terminal HPt domain (P1) from the chemotaxis kinase CheA of Thermotoga maritima reveals a remarkable degree of structural heterogeneity within a four-helix bundle. Two of the four helices have alternate main-chain conformations that differ by a 1.3-1.7A shift along the bundle axis. These dual conformers were only resolved with atomic resolution diffraction data and their inclusion significantly improved refinement statistics. Neither conformer optimizes packing within the helical core, consistent with their nearly equal refined occupancies. Altered hydrogen bonding within an inter-helical loop may facilitate transition between conformers. Two discrete structural states rather than a continuum of closely related conformations indicates an energetic barrier to conversion between conformers in the crystal at 100K, although many more states are expected in solution at physiological temperatures. Anisotropic atomic thermal B factors within the two conformers indicate modest overall atomic displacement that is largest perpendicular to the helical bundle and not along the direction of apparent motion. Despite the conformational heterogeneity of P1 in the crystal at low temperature, the protein displays high thermal stability in solution (T(m)=100 degrees C). Addition of a variable C-terminal region that corresponds to a mobile helix in other CheA structures significantly narrows the temperature width of the unfolding transition and may affect domain dynamics. Helices that compose the kinase recognition site and contain the phospho-accepting His45 do not have alternate conformations. In this region, atomic resolution provides detailed structural parameters for a conserved hydrogen-bonding network that tunes the reactivity of His45. A neighboring glutamate (E67), essential for phosphotransferase activity hydrogen bonds directly to His45 N(delta1). E67 generates a negative electrostatic surface surrounding the reactive His that is conserved by most CheA kinases, but absent in related phosphotransferase proteins. The P1 conformations that we observe are likely relevant to other helical or coiled-coil proteins and may be important for generating switches in signaling processes.

Bacterial Proteins↗

PubMed1981

A novel phosphagen phosphotransferase in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea).

Neither phosphagens nor phosphagen phosphotransferase activity could be detected in Fasciola hepatica, Hymenolepis diminuta, Moniezia expansa or in the plerocercoids of Ligula intestinalis. The plerocercoids of Schistocephalus solidus, however, possess an active taurocyamine phosphotransferase, although it too contains no detectable phosphagens. The taurocyamine phosphotransferase of S. solidus has an absolute requirement for a divalent metal ion and ATP could not be replaced by ITP, GTP, CTP or UTP as the phosphate donor. The role of phosphagens in helminths is discussed.

Adenosine Triphosphate↗

PubMed1979

Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. Copurification of HPr and alpha 1-6 glucan.

A rapid, high-yield procedure has been developed for the purification of HPr from the Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. During this procedure, the protein copurifies with a 2500-dalton homopolysaccharide which we have identified as alpha 1-6 glucan. The results of steady-state kinetic measurements of the phosphotransferase activity demonstrate that the polysaccharide works as an activator of the phosphotransferase system probably at the level of the HPr:P-E1 complex or the P-HPr:E11 complex.

Bacterial Proteins↗

PubMed1977

Purification and initial rate kinetics of acyl-phosphate-hexose phosphotransferase from Aerobacter aerogenes.

The enzyme acyl-phosphate-hexose phosphotransferase from Aerobacter aerogenes was purified to electrophoretic homogeneity. The molecular weight of the enzyme as determined on Sephadex gels is 150 000. The enzyme possesses potent phosphotransferase and phosphohydrolase activities. Initial rate kinetics were used to investigate the mechanism of acyl-phosphate-hexose phosphotransferase. These studies, which involved a number of different phosphoryl donors and substrate analogues, suggest that the kinetic mechanism is of the rapid equilibrium random Bi Bi type. A number of other enzymes that exhibit both transferase and hydrolase activities involve obligatory covalent enzyme-substrate intermediates in their mechanisms of action.

Enterobacter↗

PubMed1998

The phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius. Functional and biochemical characterization of IIABL(Man) and IIABH(Man).

Previous studies have suggested that the phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius consists of a nonphosphorylated enzyme II domain that functions in tandem with a separate enzymatic complex called III(Man). The III(Man) complex is believed to be composed of two protein dimers with molecular masses of approximately 72 kDa. Analysis of these proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has indicated that one dimer is composed of two 38.9-kDa subunits called IIIH(Man), and the other of two 35.2-kDa subunits called IIIL(Man). This study was undertaken to determine (1) the number and nature of the phosphorylated residue(s) on IIIH(Man) and IIIL(Man) and the phosphorylation sequence allowing the transfer of the phosphoryl group from HPr(His approximately P) to the mannose:PTS substrates; (2) whether IIIH(Man) and IIIL(Man) originate from two different genes or result from a posttranslational modification; and (3) whether these two proteins are involved in the phosphorylation of 2-deoxyglucose, a substrate of the phosphoenolpyruvate:mannose phosphotransferase system. We showed that both IIIH(Man) and IIIL(Man) were phosphorylated on two histidine residues. One phosphate bond was heat-labile (phosphorylation at the N1 position of the imidazole ring), while the second was heat-resistant (phosphorylation at the N3 position of the imidazole ring). The sequence of the first phosphorylation site was deduced by comparing the N-terminal amino acid sequence of both forms of III(Man) with IIA domains of the EII-mannose family. The sequences of both forms were identical over the 15 first amino acids, that is, MIGIIIASHGKFAEG. The sequence of the second phosphorylation site was determined for IIIL(Man) as IHGQVATNxTP. Hence, IIIH(Man) and IIIL(Man) are PTS proteins of the IIAB type and should be renamed IIABH(Man) and IIABL(Man). IIABH(Man) and IIABL(Man) had different peptide profiles after digestion with proteases, indicating that these two proteins are encoded by two different genes. In vitro PEP-dependent phosphorylation assays conducted with a spontaneous mutant devoid of both forms of IIAB(Man) suggested that the phosphoenolpyruvate:mannose phosphotransferase system of S. salivarius is composed of an uncharacterized nonphosphorylated membrane component that works in tandem with IIABL(Man). The physiological functions of IIABH(Man) remain unknown.

Amino Acid Sequence↗

PubMed1986

Substrate specificity and regulation of the maize (Zea mays) leaf ADP: protein phosphotransferase catalysing phosphorylation/inactivation of pyruvate, orthophosphate dikinase.

The protein substrate specificity of the maize (Zea mays) leaf ADP: protein phosphotransferase (regulatory protein, RP) was studied in terms of its relative ability to inactivate/phosphorylate pyruvate, orthophosphate dikinase from Zea mays and the non-sulphur purple photosynthetic bacterium Rhodospirillum rubrum. The dimeric bacterial dikinase was inactivated by the maize leaf RP via phosphorylation, with a stoichiometry of approximately 1 mol of phosphate incorporated/mol of 92.7-kDa protomer. Inactivation required both ADP and ATP, with ADP being the specific donor for regulatory phosphorylation. The requirements for inactivation/phosphorylation in this heterologous system were identical with those previously established for the tetrameric maize leaf dikinase. The ADP-dependent maize leaf RP did not phosphorylate alternative protein substrates such as casein or phosvitin, and its activity was not affected by cyclic nucleotides, Ca2+ or calmodulin. The regulation of the maize leaf ADP: protein phosphotransferase was studied in terms of changes in adenylate energy charge and pyruvate concentration. The change in adenylate energy charge necessary to substantially inhibit phosphorylation of maize leaf dikinase was not suggestive of it being a physiological modulator of phosphotransferase activity. Pyruvate was a potent competitive inhibitor of regulatory phosphorylation (Ki = 80 microM), consistent with its interaction with the catalytic phosphorylated intermediate of dikinase, the true protein substrate for ADP-dependent phosphorylation/inactivation.

Adenosine Diphosphate↗

PubMed1987

Properties of N-acetylglucosamine 1-phosphotransferase from human lymphoblasts.

Human lymphoblast and fibroblast cell lines from a patient with I-cell disease and normal individuals were characterized with respect to certain properties of UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine phosphotransferase. The enzyme isolated from normal lymphoblast and fibroblast cell lines expressed similar kinetic properties, substrate specificities and subcellular localizations. Coincident with the severe reduction of N-acetylglucosamine phosphotransferase activity in both I-cell fibroblast and lymphoblast cell lines, there was an increased secretion of several lysosomal enzymes compared to normal controls. Subsequent examination of N-acetyl-beta-D-hexosaminidase secreted by the I-cell lymphoblasts demonstrated a significant increase in adsorption of the I-cell enzyme to Ricinus communis agglutinin, a galactose-specific lectin. However, the I-cell lymphoblasts did not exhibit the significant decrease in intracellular lysosomal activities seen in I-cell fibroblasts. Our results suggest that lymphoblasts not only represent an excellent source for the purification of N-acetylglucosamine phosphotransferase, but in addition, represent a unique system for studying alternate mechanisms involved in the targeting of lysosomal enzymes.

Cell Line↗

PubMed1982

Novel sensory adaptation mechanism in bacterial chemotaxis to oxygen and phosphotransferase substrates.

The involvement of methylation in the chemosensory response of bacteria to many attractants has been clearly established by studies in several laboratories. It has been assumed that adaptation of Salmonella typhimurium and Escherichia coli to all attractants involves methylation of a transmembrane methyl-accepting chemotaxis protein. The methyl donor in this reaction is S-adenosyl-L-methionine, and the protein methyltransferase is the product of the cheR gene. In contrast, adaptation to oxygen and phosphotransferase substrates were found to be independent of this methylation system. In E. coli AW660 (tsr tar trg), which lacks the known methyl-accepting chemotaxis proteins, chemotaxis was normal to oxygen and to substrates of the phosphotransferase system such as D-mannose, D-glucose, and N-acetyl-D-glucosamine. When S-adenosyl-L-methionine was depleted by methionine starvation or by addition of 1-aminocyclopentane-1-carboxylic acid, methylation-dependent adaptation to serine, aspartate, and ribose was defective in wild-type E. coli and S. typhimurium. However, adaptation to oxygen and phosphotransferase substrates was independent of S-adenosyl-L-methionine and the cheR product. These results suggest that there are methylation-independent and methylation-dependent mechanisms for sensory adaptation in bacteria.

Acclimatization↗