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PubMed1982

Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a phosphocarrier protein HPr from wild type and mutants of Salmonella typhimurium.

HPr, the histidine-containing phosphocarrier protein of the phosphotransferase system, has been isolated and purified from wild type Salmonella typhimurium and from two mutants of this organism. Comparison of the sequences of these three forms of HPr indicates that the two mutants contain substitutions at residue 4 in the polypeptide chain; in place of glutamine in the wild type, one mutant (SB3899) contained serine and the other (SB3093) contained lysine. The substitution of lysine for glutamine resulted in increased positive charge of the molecule which is reflected in an expected decreased mobility on polyacrylamide gel electrophoresis and its behavior on isoelectric focusing. However, these changes had no effect on phosphocarrier activity in the phoshoenolpyruvate:glycose phosphotransferase system as measured by the kinetics of the interaction on the mutant HPr proteins with both Enzyme I and Enzyme II. These results have important implications for potential chemical modification of HPr. The HPr from wild type cells was crystallized.

Amino Acid Sequence↗

PubMed1982

Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a glucose-specific phosphocarrier protein (IIIGlc) from Salmonella typhimurium.

The phosphocarrier protein, IIIGlc, of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was purified to homogeneity by two methods. The first method utilized ion exchange and gel filtration chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis, and required several weeks for completion. The second method utilized and antibody affinity column plus two additional steps and could be completed in a few days. By both procedures, two forms of IIIGlc were isolated, which were called IIIGlc Slow and IIIGlc Fast on the basis of their relative mobilities in polyacrylamide gels. IIIGlc Fast is derived from IIIGlc Slow by cleavage of the seven NH2-terminal amino acids from the latter protein. Both IIIGlc Slow and IIIGlc Fast have Mr approximately 20,000; neither protein contains cysteine, tyrosine, or tryptophan. IIIGlc Slow is very stable to heat; only 50% of its sugar phosphorylating activity is lost after 1 h at 100 degrees C. The phosphoryl group in IIIGlc Slow appears to be linked to a histidinyl residue. Direct transfer of the phosphoryl group from HPr (the histidine-containing phosphocarrier protein of the PTS) to IIIGlc slow was demonstrated as well as the reverse reaction. In addition, phospho-IIIGlc Slow served as a phosphoryl donor to methyl alpha-glucoside (or glucose) in the absence of all other PTS components except the partially purified integral membrane protein specific for this sugar, II-BGlc. The loss of the seven amino acids from IIIGlc Slow (giving IIIGlc Fast) leads to a marked alteration in the kinetic properties of the protein in the phosphotransferase system. IIIGlc Slow accepts 1 mol of phosphate from phosphoenolpyruvate via Enzyme I and HPr (the histidine-containing phosphocarrier protein) and participates in the phosphorylation of glucose or methyl alpha-D-glucoside. IIIGlc Fast also accepts 1 mol of phosphate, but phospho-IIIGlc Fast is only 2-3% as active as phospho-IIIGlc Slow in the phosphorylation of sugar. IIIGlc Fast is found only in trace quantities in living cells, and may play a role in the regulation of non-PTS sugar transport systems.

Amino Acid Sequence↗

PubMed1982

Sugar transport by the bacterial phosphotransferase system. Preparation and characterization of membrane vesicles from mutant and wild type Salmonella typhimurium.

Modifications of published procedures (reviewed by Kaback, H. R. (1974) Science (Wash. D. C.) 186, 882-892) were developed for preparing membrane vesicles from Salmonella typhimurium. The preparations consisted largely of closed, unilamellar structures and contained inner membrane with little to no contamination by outer membrane or cell wall. A variety of cytoplasmic proteins was assayed in the membrane preparations, and they were found to be present at low to trace levels, whereas other proteins known to be associated with membranes were found at high levels (with respect to specific activities) in the vesicle preparations. At least 90% of the vesicles appeared to be oriented right-side-out; we do not know whether the remaining 10% represents closed vesicles oriented inside-out or "leaky" right-side-out vesicles. The vesicle preparations were impermeable to both low and high molecular weight solutes, for example, to both intra- and extravesicular sucrose. In double label experiments, the vesicle volumes were found to be about 6 microliters/mg of protein for preparations isolated from the wild type strain, and about 4.5 microliters/mg of protein for vesicles isolated from a mutant, SB2950, deleted in ptsH, ptsI, and crr genes (proteins HPr, Enzyme I, and IIIGlc, respectively). One advantage of S. typhimurium over Escherichia coli for these studies is that the former can be induced to take up phosphoenolpyruvate. This may be the reason that S. typhimurium vesicles transported methyl alpha-glucoside at 4- to 100-fold the rates reported for vesicles from E. coli, while uptake rates of proline were comparable in the two types of preparations. Vesicles from strain SB2950 were unable to take up methyl alpha-glucoside, but the transport (and phosphorylating) system was reconstituted in the vesicles by trapping the soluble purified proteins inside the vesicles during preparation of the latter. All three proteins were required for reconstruction. Studies with intra- and extravesicular soluble proteins of the phosphoenolpyruvate:glucose phosphotransferase system showed that the IIMan complex, which phosphorylates glucose, 2-deoxyglucose, and other sugars, is symmetrically oriented in the membranes. That is, this complex could phosphorylate 2-deoxyglucose when supplemented with Enzyme I and HPr either inside or outside of the membranes, and the sugar phosphate was found on the same side of the membranes as the soluble phosphotransferase system proteins. The integral membrane protein, II-BGlc, which phosphorylates glucose and methyl alpha-glucoside, showed contrasting behavior. Methyl alpha-glucoside phosphate was formed (intravesicularly) only when the soluble proteins (Enzyme I, HPr, and IIIGlc) were located inside the vesicles. Thus, II-BGlc appears to be asymmetrically oriented in the membranes.

Biological Transport↗

PubMed1976

Direct transfer of the phosphoryl moiety of mannitol 1-phosphate to [14C]mannitol catalyzed by the enzyme II complexes of the phosphoenolpyruvate: mannitol phosphotransferase systems in Spirochaeta aurantia and Salmonella typhimurium.

Spirochaeta aurantia possesses a phosphoenolpyruvate:mannitol phosphotransferase system which catalyzes the transmembrane transport and phosphorylation of mannitol. In vitro studies showed that both phosphoenolpyruvate and mannitol 1-phosphate could serve as phosphate donors. The phosphoenolpyruvate-dependent reaction required two soluble proteins, Enzyme SI and HPr, and an integral membrane complex, Enzyme SII. Only Enzyme SII was required for the mannitol 1-phosphate-dependent reaction. Enzyme II-dependent transphosphorylation of sugars was also demonstrated in eubacterial extracts. The results lead to the suggestion that the Enzyme II complexes of bacterial phosphotransferase systems possess nonoverlapping binding sites for sugar and sugar phosphate.

Edetic Acid↗

PubMed1993

Deamidation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system, involves asparagine 38 (HPr-1) and asparagine 12 (HPr-2) in isoaspartyl acid formation.

Histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli, when incubated at elevated temperatures forms many species of protein. The two major species are HPr-1 and HPr-2, which have been shown to lack one or two amides, respectively (Anderson, B., Weigel, N., Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 7023-7033). The formation of HPr-1 and HPr-2 is shown to be pH-dependent and does not occur readily below pH 6. Investigation of the identities and properties of the two residues that deamidate involved creation of site-directed mutants at the 6 glutamine and 2 asparagine residues of HPr; description of their deamidation species by isoelectric focusing; determination of their relative antibody binding properties; assay of their phosphoacceptor and phosphodonor activities; characterization of tryptic and V8-protease peptides; obtaining two-dimensional nuclear magnetic resonance spectra of HPr, HPr-1, and several mutants. It was determined that the sequential deamidation of Asn-38 and Asn-12 yields HPr-1 and HPr-2. Both residues exist as Asn-Gly pairs, and both deamidations probably form isoaspartyl acid. HPr from Bacillus subtilis and Staphylococcus carnosus which also have Asn-Gly at residues 38 and 39 form HPr-1 species presumably by deamidation. HPr from Streptococcus faecalis which does not have Asn-38 does not form a HPr-1 species. The E. coli mutant HPrs, N12D and Q51E, residues that may be involved in the active site, had impaired phosphohydrolysis properties and decreased phosphoenolpyruvate:sugar phosphotransferase system activity.

Amino Acid Sequence↗

PubMed1997

A conditional lethal yeast phosphotransferase (tpt1) mutant accumulates tRNAs with a 2'-phosphate and an undermodified base at the splice junction.

tRNA splicing is essential in yeast and humans and presumably all eukaryotes. The first two steps of yeast tRNA splicing, excision of the intron by endonuclease and joining of the exons by tRNA ligase, leave a splice junction bearing a 2'-phosphate. Biochemical analysis suggests that removal of this phosphate in yeast is catalyzed by a highly specific 2'-phosphotransferase that transfers the phosphate to NAD to form ADP-ribose 1"-2" cyclic phosphate. 2'-Phosphotransferase catalytic activity is encoded by a single essential gene, TPT1, in the yeast Saccharomyces cerevisiae. We show here that Tpt1 protein is responsible for the dephosphorylation step of tRNA splicing in vivo because, during nonpermissive growth, conditional lethal tpt1 mutants accumulate 2'-phosphorylated tRNAs from eight different tRNA species that are known to be spliced. We show also that several of these tRNAs are undermodified at the splice junction residue, which is always located at the hypermodified position one base 3' of the anticodon. This result is consistent with previous results indicating that modification of the hypermodified position occurs after intron excision in the tRNA processing pathway, and implies that modification normally follows the dephosphorylation step of tRNA splicing in vivo.

Genes, Fungal↗

PubMed1986

Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase system.

Recently we reported the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococcus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system (J. Deutscher, FEMS Microbiol. Lett. 29:237-243, 1985). The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The Kms were found to be 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 mM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitive manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following [32P]PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, we isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.

Bacterial Proteins↗

PubMed2005

A splicing mutation in the alpha/beta GlcNAc-1-phosphotransferase gene results in an adult onset form of mucolipidosis III associated with sensory neuropathy and cardiomyopathy.

A 47-year-old female who presented with a dilated cardiomyopathy and mild neuropathy was found to have pseudoHurler polydystrophy (mucolipidosis III). The serum lysosomal enzymes were strikingly elevated and GlcNAc-1-phosphotransferase activity in the patient's fibroblasts was 3% of normal. Sequence analysis of the patient's genomic DNA revealed a homozygous mutation of the last nucleotide of the 135-bp exon 7 of the phosphotransferase gene encoding the alpha/beta subunits, resulting in aberrant splicing and skipping of this exon. Remarkably, none of the skeletal and connective tissue anomalies characteristic of the disease were present. This case is the first example of mucolipidosis III presenting in an adult patient and further broadens the clinical spectrum of the disease.

Age of Onset↗

PubMed2005

Solution structure and dynamics of LuxU from Vibrio harveyi, a phosphotransferase protein involved in bacterial quorum sensing.

The marine bacterium Vibrio harveyi controls its bioluminescence by a process known as quorum sensing. In this process, autoinducer molecules are detected by membrane-bound sensor kinase/response regulator proteins (LuxN and LuxQ) that relay a signal via a series of protein phosphorylation reactions to another response regulator protein, LuxO. Phosphorylated LuxO indirectly represses the expression of the proteins responsible for bioluminescence. Integral to this quorum sensing process is the function of the phosphotransferase protein, LuxU. LuxU acts to shuttle the phosphate from the membrane-bound proteins, LuxN and LuxQ, to LuxO. LuxU is a 114 amino acid residue monomeric protein. Solution NMR was used to determine the three-dimensional structure of LuxU. LuxU contains a four-helix bundle topology with the active-site histidine residue (His58) located on alpha-helix C and exposed to solution. The active site represents a cluster of positively charged residues located on an otherwise hydrophobic protein face. NMR spin-relaxation experiments identify a collection of flexible residues localized on the same region of LuxU as His58. The studies described here represent the first structural characterization of an isolated, monomeric bacterial phosphotransferase protein.

Amino Acid Sequence↗

PubMed2003

Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca(2+)-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P). Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP. Cosolvents [isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)] also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles. Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates. PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T(m) for PI-PLC thermal denaturation decreased linearly with added iPrOH). However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein. At 30% iPrOH and 4 degrees C (well below the T(m) for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal. iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate. This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure.

Bacillus thuringiensis↗

PubMed1994

Escherichia coli glycerol kinase: role of a tetramer interface in regulation by fructose 1,6-bisphosphate and phosphotransferase system regulatory protein IIIglc.

Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) is a key element in a signal transduction pathway that couples expression of genes required for glycerol metabolism to the relative availability of glycerol and glucose. Its catalytic activity is inhibited by protein-protein interactions with IIIglc, a phosphotransferase system protein, and by fructose 1,6-bisphosphate (FBP); each of these allosteric effectors constitutes a positive signal that glucose is available. Loss of glucose inhibition of glycerol metabolism was used to screen for regulatory mutants of glycerol kinase after hydroxylamine mutagenesis of the cloned glpK gene. Two mutant enzymes were identified and shown by DNA sequencing to contain the mutations alanine 65 to threonine (A65T) and aspartate 72 to asparagine (D72N). Initial velocity studies show the mutations do not significantly affect the catalytic properties, hence active-site structures, of the enzymes. Both mutations decrease inhibition by FBP; A65T eliminates the inhibition while D72N appears to decrease the affinity for FBP and the extent of the inhibition. However, neither mutation significantly affects inhibition by IIIglc. Gel-permeation chromatography studies show that both of the mutations alter the dimer-tetramer assembly reaction of the enzyme and the effect of FBP in increasing the molecular weight. The effects of the mutations on the assembly reaction are consistent with the locations of these two amino acid residues in the X-ray structure, which shows them to be associated with an alpha-helix that constitutes one of the two subunit-subunit interfaces within the tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1985

Amino acid sequence of the amphiphilic phosphocarrier protein factor IIILac of the lactose-specific phosphotransferase system of Staphylococcus.

The lactose-specific factor III of the phosphotransferase system of Staphylococcus aureus is an amphiphilic trimeric protein composed of identical subunits. It is hydrophilic in its unphosphorylated state and can be isolated from the cytoplasmic protein fraction. It becomes a constituent of the membrane-bound phosphotransferase complex upon phosphorylation of a single histidyl residue. The sequence of S. aureus factor IIILac was determined and revealed that the subunits consist of 103 residues corresponding to a Mr of 11 367 and of 34 101 for the native trimer: (sequence; see text) According to this sequence and previous work histidine residue 82 located in the C-terminal part of the polypeptide chain is phosphorylated at the N-3 position by phosphoenolpyruvate, enzyme I, and histidine-containing phosphocarrier protein. The N-terminal part of the protein comprising approximately one-third of the chain exhibits in vitro affinity toward membrane-bound enzyme IILac.

Amino Acid Sequence↗

PubMed2004

Thermodynamics of aminoglycoside binding to aminoglycoside-3'-phosphotransferase IIIa studied by isothermal titration calorimetry.

The aminoglycoside-3'-phosphotransferase IIIa [APH(3')-IIIa] phosphorylates aminoglycoside antibiotics and renders them ineffective against bacteria. APH(3')-IIIa is the most promiscuous aminoglycoside phosphotransferase enzyme, and it modifies more than 10 different aminoglycoside antibiotics. A wealth of information exists about the enzyme; however, thermodynamic properties of enzyme-aminoglycoside complexes are still not known. This study describes the determination of the thermodynamic parameters of the binary enzyme-aminoglycoside and the ternary enzyme-metal-ATP-aminoglycoside complexes of structurally related aminoglycosides using isothermal titration calorimetry. Formation of the binary enzyme-aminoglycoside complexes is enthalpically driven and exhibits a strongly disfavored entropic contribution. Formation of the ternary enzyme-metal-ATP-aminoglycoside complexes yields much smaller negative DeltaH values and more favorable entropic contributions. The presence of metal-ATP generally increases the affinity of aminoglycosides to the enzyme. This is consistent with the kinetic mechanism of the enzyme in which ordered binding of substrates occurs. However, the observed DeltaH values neither correlate with kinetic parameters k(cat), K(m), and k(cat)/K(m) nor correlate with the molecular size of the substrates. Comparison of the thermodynamic properties of the complexes formed by structurally similar aminoglycosides indicated that the 2'- and the 6'-amino groups of the substrates are involved in binding to the enzyme. Thermodynamic properties of the complexes formed by aminoglycosides differing only at the 3'-hydroxyl group suggested that the absence of this group does not alter the thermodynamic parameters of the ternary APH(3')-IIIa-metal-ATP-aminoglycoside complex. Our results also indicate that protonation of ligand and protein ionizable groups is coupled to the complex formation between aminoglycosides and APH(3')-IIIa. Comparison of DeltaH values for different aminoglycoside-enzyme complexes indicates that enzyme and substrates undergo significant conformational changes in complex formation.

Adenosine Triphosphate↗

PubMed2002

Activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase by fructose 2,6-bisphosphate stimulates conversion of hexose phosphates to triose phosphates but does not influence accumulation of carbohydrates in phosphate-deficient tobacco cells.

The aim of this work was to investigate the contribution of fructose 2,6-bisphosphate to the regulation of carbohydrate metabolism under phosphate stress. The study exploited heterotrophic tobacco callus lines expressing a modified mammalian 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase that increased the fructose 2,6-bisphosphate content of the tissue. The phosphate status of two transgenic and one untransformed cell line was perturbed by incubation with 2-deoxyglucose, a phosphate-sequestering agent, and by growth of callus on phosphate-depleted media. 31P-NMR spectroscopy confirmed that both treatments decreased cellular levels of inorganic phosphate and phosphorylated metabolites. Despite large decreases in the amounts of phosphate esters, UDPglucose and adenylates in response to phosphate deficiency, the fructose 2,6-bisphosphate content of each line was unaffected by 2-deoxyglucose and increased during growth on phosphate-limited media. Short-term treatment of callus with 2-deoxyglucose had only minor effects on the carbohydrate status of each line, whereas long-term phosphate deficiency caused an increase in starch and a decrease in soluble sugar content in both transgenic and control lines. There were no consistent differences between the three callus lines in metabolism of [U-14C]glucose in response to incubation with 2-deoxyglucose. In contrast, there was a decrease in partitioning of label into glycolytic products (particularly organic acids) in untransformed callus during growth on phosphate-depleted medium. This decrease was greatly attenuated in the transgenic lines with increased fructose 2,6-bisphosphate content. This suggests that the conversion of hexose phosphates to triose phosphates is constrained under phosphate-deficient conditions, and that this restriction can be relieved by activation of pyrophosphate:fructose-6-phosphate 1-phosphotransferase. However, since the transgenic and control lines did not differ in the extent to which the carbohydrate content changed in response to growth on phosphate-depleted media, it is concluded that an increase in flux through pyrophosphate:fructose-6-phosphate 1-phosphotransferase is not a major component of the metabolic response of heterotrophic tobacco cells to phosphate deficiency.

Journal Article↗

PubMed1983

Partial characterization of the phosphotransferase system of human central-nervous-system myelin.

The phosphotransferase system of human central-nervous-system myelin was investigated. Evidence obtained indicated the presence of at least two different phosphotransferase systems (cyclic nucleotide-dependent and -independent) in myelin, which were found to be firmly associated with the membrane. The cyclic AMP-dependent kinase of myelin and white-matter cytosol preferentially phosphorylated certain histone fractions and displayed only modest activity with basic protein as substrate. On the other hand, the cyclic nucleotide-independent system showed specificity toward basic protein. Its activity was not only dependent on Mg2+ but it was greatly enhanced by this bivalent cation. Whereas the cyclic nucleotide-dependent kinase could be extracted with buffers containing Triton X-100, the bivalent cation-regulated kinase resisted solubilization from myelin under these conditions.

Brain↗

PubMed2003

Soluble sugar permeases of the phosphotransferase system in Escherichia coli: evidence for two physically distinct forms of the proteins in vivo.

The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) consists of a set of cytoplasmic energy-coupling proteins and various integral membrane permeases/sugar phosphotransferases, each specific for a different sugar. We have conducted biochemical analyses of three PTS permeases (enzymes II), the glucose permease (IIGlc), the mannitol permease (IIMtl) and the mannose permease (IIMan). These enzymes each catalyse two vectorial/chemical reactions, sugar phosphorylation using phosphoenolpyruvate (PEP) as the phosphoryl donor, dependent on enzyme I, HPr and IIA as well as IIBC (the PEP reaction), and transphosphorylation using a sugar phosphate (glucose-6-P for IIGlc and IIMan; mannitol-1-P for IIMtl) as the phosphoryl donor, dependent only on IIBC (the TP reaction). When crude extracts of French-pressed or osmotically shocked Escherichia coli cells are centrifuged in an ultracentrifuge at high speed, 5-20% of the enzyme II activity remains in the high-speed supernatant, and passage through a gel filtration column gives two activity peaks, one in the void volume exhibiting high PEP-dependent and TP activities, and a second included peak with high PEP-dependent activity and high (IIMan), moderate (IIGlc) or negligible (IIMtl) TP activities. Both log and stationary phase cells exhibit comparable relative amounts of pelletable and soluble enzyme II activities, but long-term exposure of cells to chloramphenicol results in selective loss of the soluble fraction with retention of much of the pelleted activity concomitant with extensive protein degradation. Short-term exposure of cells to chloramphenicol results in increased activities in both fractions, possibly because of increased lipid association, with more activation in the soluble fraction than in the pelleted fraction. Western blot analyses show that the soluble IIGlc exhibits a subunit size of about 45 kDa, and all three soluble enzymes II elute from the gel filtration column with apparent molecular weights of 40-50 kDa. We propose that enzymes II of the PTS exist in two physically distinct forms in the E. coli cell, one tightly integrated into the membrane and one either soluble or loosely associated with the membrane. We also propose that the membrane-integrated enzymes II are largely dimeric, whereas the soluble enzymes II, retarded during passage through a gel filtration column, are largely monomeric.

Blotting, Western↗

PubMed1995

A novel mutagenesis strategy identifies distantly spaced amino acid sequences that are required for the phosphorylation of both the oligosaccharides of procathepsin D by N-acetylglucosamine 1-phosphotransferase.

A novel combinatorial mutagenesis strategy (shuffle mutagenesis) was developed to identify sequences in the propiece and amino lobe of cathepsin D which direct oligosaccharide phosphorylation by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. Propiece restriction fragments and oligonucleotide cassettes corresponding to 13 regions of the cathepsin D and glycopepsinogen amino lobes were randomly shuffled together to generate a large library of chimeric molecules. The library was inserted into an expression vector encoding the carboxyl lobe of cathepsin D with a carboxyl-terminal myc epitope and a CD8 transmembrane extension. Transfected COS1 cells expressing the membrane-anchored forms of the cathepsin D/glycopepsinogen chimeras at the cell surface were selected with solid phase mannose 6-phosphate receptor or an antibody to the myc epitope. Plasmids were rescued in Escherichia coli and sequenced by hybridization to the original oligonucleotide cassettes. Two regions of the cathepsin D amino lobe (segments 7 and 12) were found to contribute to proper folding, surface expression, and selective phosphorylation of the carboxyl lobe oligosaccharide. Two different cathepsin D regions (the propiece and segment 5) cooperated with a previously identified recognition element in the carboxyl lobe to allow efficient phosphorylation of both the amino and carboxyl lobe oligosaccharides. Three general models for extending the catalytic reach of N-acetylglucosamine 1-phosphotransferase to widely spaced oligosaccharides are presented.

Amino Acid Sequence↗

PubMed1998

Competitive inhibition of choline phosphotransferase by geranylgeraniol and farnesol inhibits phosphatidylcholine synthesis and induces apoptosis in human lung adenocarcinoma A549 cells.

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoid-induced apoptosis was preceded by an arrest in G0/G1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.

Actins↗