SEARCH · PubMed Health

Results for “PHOSPHOTRANSFERASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources

PubMed1982

Phosphoenolpyruvate-dependent fructose phosphotransferase system of Rhodopseudomonas sphaeroides: purification and physicochemical and immunochemical characterization of a membrane-associated enzyme I.

The phosphotransferase system (PTS) of the phototrophic bacterium Rhodopseudomonas sphaeroides consists of a component located in the cytoplasmic membrane and a membrane-associated enzyme called "soluble factor" (SF) [Saier, M. H., Feucht, B. U., & Roseman, S. (1971) J. Biol. Chem. 246, 7819--7821]. SF has been partially purified by a combination of hydrophobic interaction and ion-exchange and gel-permeation chromatography. SF is similar to Escherichia coli enzyme I in its molecular characteristics and enzymatic properties. It has a molecular weight of 85 000 and readily dimerizes. Phosphoenolpyruvate and Mg2+ stabilize the dimer. The enzyme catalyzes the conversion of phosphoenolpyruvate into pyruvate and becomes phosphorylated in the process. The phosphoryl group is subsequently transferred to fructose in the presence of R. sphaeroides membranes. SF substitutes for E. coli enzyme I in fructose or glucose phosphorylation with E. coli enzyme II and HPr. The activities of SF with the R. sphaeroides PTS and the E. coli PTS reside on structurally distinct molecules as shown by their response to limited proteolytic digestion and by immunochemical studies. The activity of SF with the E. coli PTS arises during the isolation procedure and is most likely due to the removal of HPr-like protein from SF.

Chromatography↗

PubMed1979

Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. NMR studies of the conformation of HPr and P-HPr and the mechanism of energy coupling.

1H and 31P nuclear magnetic resonance investigations of the phosphoprotein intermediate P-HPr and the parent molecule HPr of the E. coli phosphoenolpyruvate dependent phosphotransferase system (PTS) show that HPr can exist in two conformations. These conformations influence the protonation state of the reactive histidine residue, thereby determining the reaction pathway in the phosphoryl group transfer step. A general mechanism is proposed for the energy-coupling process in the PTS.

Bacterial Proteins↗

PubMed2001

Substrate recognition by a yeast 2'-phosphotransferase involved in tRNA splicing and by its Escherichia coli homolog.

The final step of tRNA splicing in Saccharomyces cerevisiae requires 2'-phosphotransferase (Tpt1) to transfer the 2'-phosphate from ligated tRNA to NAD, producing mature tRNA and ADP ribose-1' '-2' '-cyclic phosphate. To address how Tpt1 protein recognizes substrate RNAs, we measured the steady-state kinetic parameters of Tpt1 protein with 2'-phosphorylated ligated tRNA and a variety of related substrates. Tpt1 protein has a high apparent affinity for ligated tRNA (K(m,RNA), 0.35 nM) and a low turnover rate (k(cat), 0.3 min(-1)). Tpt1 protein recognizes both tRNA and the internal 2'-phosphate of RNAs. Steady-state kinetic analysis reveals that as RNAs lose structure and length, K(m,RNA) and k(cat) both increase commensurately. For a 2'-phosphorylated octadecamer derived from the anticodon stem-loop of ligated tRNA, K(m,RNA) and k(cat) are 5- and 8-fold higher, respectively, than for ligated tRNA, whereas for a simple substrate like pApA(p)pA, K(m,RNA) and k(cat) are 430- and 150-fold higher, respectively. Tpt1 is not detectably active on a trimer with a terminal 5'- or 3'-phosphate and is very inefficient at removal of a terminal 2'-phosphate unless there is an adjacent 3'-phosphate or phosphodiester. The K(m,NAD) for Tpt1 is substrate dependent: K(m,NAD) is 10 microM with ligated tRNA, 200 microM with pApA(p)pA, and 600 microM with pApApA(p). Preliminary analysis of KptA, a functional Tpt1 protein homologue from Escherichia coli, reveals that KptA protein is strikingly similar to yeast Tpt1 in its kinetic parameters, although E. coli is not known to have a 2'-phosphorylated RNA substrate.

Escherichia coli↗

PubMed2002

Interdomain interaction and substrate coupling effects on dimerization and conformational stability of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The bacterial PEP:sugar phosphotransferase system couples the phosphorylation and translocation of specific sugars across the membrane. The activity of the first protein in this pathway, enzyme I (EI), is regulated by a monomer-dimer equilibrium where a Mg(2+)-dependent autophosphorylation by PEP requires the dimer. Dimerization constants for dephospho- and phospho-EI and inactive mutants EI(H189E) and EI(H189A) (in which Glu or Ala is substituted for the active site His189) have been measured under a variety of conditions by sedimentation equilibrium at pH 7.5 and 4 and 20 degrees C. Concurrently, thermal unfolding of these forms of EI has been monitored by differential scanning calorimetry and by changes in the intrinsic tryptophanyl residue fluorescence. Phosphorylated EI and EI(H189E) have 10-fold increased dimerization constants [ approximately 2 x 10(6) (M monomer)(-1)] compared to those of dephospho-EI and EI(H189A) at 20 degrees C. Dimerization is strongly promoted by 1 mM PEP with 2 mM MgCl(2) [K(A)' > or = 10(8) M(-1) at 4 or 20 degrees C], as demonstrated with EI(H189A) which cannot undergo autophosphorylation. Together, 1 mM PEP and 2 mM Mg(2+) also markedly stabilize and couple the unfolding of C- and N-terminal domains of EI(H189A), increasing the transition temperature (T(m)) for unfolding the C-terminal domain by approximately 18 degrees C and that for the N-terminal domain by approximately 9 degrees C to T(max) congruent with 63 degrees C, giving a value of K(D)' congruent with 3 microM PEP at 45 degrees C. PEP alone also promotes the dimerization of EI(H189A) but only increases T(m) approximately 5 degrees C for C-terminal domain unfolding without affecting N-terminal domain unfolding, giving an estimated value of K(D)' congruent with 0.2 mM for PEP dissociation in the absence of Mg(2+) at 45 degrees C. In contrast, the dimerization constant of phospho-EI at 20 degrees C is the same in the absence and presence of 5 mM PEP and 2 mM MgCl(2). Thus, the separation of substrate binding effects from those of phosphorylation by studies with the inactive EI(H189A) has shown that intracellular concentrations of PEP and Mg(2+) are important determinants of both the conformational stability and dimerization of dephospho-EI.

Amino Acid Substitution↗

PubMed2002

Binding of enzyme IIAGlc, a component of the phosphoenolpyruvate:sugar phosphotransferase system, to the Escherichia coli lactose permease.

Enzyme IIA(Glc), encoded by the crr gene of the phosphoenolpyruvate:sugar phosphotransferase system, plays an important role in regulating intermediary metabolism in Escherichia coli ("catabolite repression"). One function involves inhibition of inducible transport systems ("inducer exclusion"), and with lactose permease, a galactoside is required for unphosphorylated IIA(Glc) binding to cytoplasmic loops IV/V and VI/VII [Sondej, M., Sun, J. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530]. With inside-out membrane vesicles containing the permease, [(125)I]IIA(Glc) binding promoted by melibiose exhibits an affinity (K(D)(IIA)) of approximately 1 microM and a stoichiometry of one mole of IIA(Glc) per six moles of lactose permease. Both the quantity of [(125)I]IIA(Glc) bound and the sugar concentration required for half-maximal IIA(Glc) binding (K(0.5)(IIA)(sug)) was measured for eight permease substrates. Differences in maximal IIA(Glc) binding are observed, and the K(0.5)(IIA)(sug) does not correlate with the affinity of LacY for sugar. Furthermore, K(0.5)(IIA)(sug) does not correlate with sugar affinities for various permease mutants. IIA(Glc) does not bind to a mutant (Cys154 --> Gly), which is locked in an outwardly facing conformation, binds with increased stoichiometry to mutant Lys131 --> Cys, and binds only weakly to two other mutants which appear to be predominantly in either an outwardly or an inwardly facing conformation. When the latter two mutations are combined, sugar-dependent IIA(Glc) binding returns to near wild-type levels. The findings suggest that binding of various substrates to lactose permease results in a collection of unique conformations, each of which presents a specific surface toward the inner face of the membrane that can interact to varying degrees with IIA(Glc).

Amino Acid Sequence↗

PubMed2006

Synthesis of HPr(Ser-P)(His-P) by enzyme I of the phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius.

HPr is a protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In Gram-positive bacteria, HPr can be phosphorylated on Ser(46) by HPr(Ser) kinase/phosphorylase (HPrK/P) and on His(15) by enzyme I (EI) of the PTS. In vitro studies have shown that phosphorylation on one residue greatly inhibits the second phosphorylation. However, streptococci contain significant amounts of HPr(Ser-P)(His approximately P) during exponential growth, and recent studies suggest that phosphorylation of HPr(Ser-P) by EI is involved in the recycling of HPr(Ser-P)(His approximately P). We report in this paper a study on the phosphorylation of Streptococcus salivarius HPr, HPr(Ser-P), and HPr(S46D) by EI. Our results indicate that (i) the specificity constant (k(cat)/K(m)) of EI for HPr(Ser-P) at pH 7.9 was approximately 5000-fold smaller than that observed for HPr, (ii) no metabolic intermediates were able to stimulate HPr(Ser-P) phosphorylation, (iii) the rate of HPr phosphorylation decreased at pHs below 6.5, while that of HPr(Ser-P) increased and was almost 10-fold higher at pH 6.1 than at pH 7.9, (iv) HPr(S46D), a mutated HPr alleged to mimic HPr(Ser-P), was also phosphorylated more efficiently under acidic conditions, and, lastly, (v) phosphorylation of Bacillus subtilis HPr(Ser-P) by B. subtilis EI was also stimulated at acidic pH. Our results suggest that the high levels of HPr(Ser-P)(His approximately P) in streptococci result from the combination of two factors, a high physiological concentration of HPr(Ser-P) and stimulation of HPr(Ser-P) phosphorylation by EI at acidic pH, an intracellular condition that occurs in response to the acidification of the external medium during growth of the culture.

Base Sequence↗

PubMed1996

Regiospecificity of aminoglycoside phosphotransferase from Enterococci and Staphylococci (APH(3')-IIIa).

The broad-spectrum aminoglycoside phosphotransferase, APH(3')-IIIa, confers resistance to several aminoglycoside antibiotics in opportunistic pathogens of the genera Staphylococcus and Enterococcus. The profile of the drug resistance phenotype suggested that the enzyme would transfer a phosphate group from ATP to the 3'-hydroxyl of aminoglycosides. In addition, resistance to the 3'-deoxyaminoglycoside antibiotic, lividomycin A, suggested possible transfer to the 5"-hydroxyl of the ribose [Trieu-Cuot, P., & Courvalin, P. (1983) Gene 23, 331-341]. Using purified overexpressed enzyme, we have prepared and purified the products of APH(3')-IIIa-dependent phosphorylation of several of aminoglycoside antibiotics. Mass spectral analysis revealed that 4,6-disubstituted aminocyclitol antibiotics such as amikacin and kanamycin are monophosphorylated, while 4,5-disubstituted aminoglycosides such as butirosin A, ribostamycin, and neomycin B are both mono- and diphosphorylated by APH(3')-IIIa. Using a series of one- and two-dimensional 1H, 13C, and 31P NMR experiments, we have unambiguously assigned the regiospecificity of phosphoryl transfer to several antibiotics. The 4,6-disubstituted aminocyclitol antibiotics are exclusively phosphorylated at the 3'-OH hydroxyl, and the 4,5-disubstituted aminocyclitol antibiotics can be phosphorylated at both the 3'- and 5"-hydroxyls. The first phosphorylation can occur on either the 3'- or 5"-hydroxyl group of neomycin B or butirosin A. Initial phosphotransfer to the 3'-position predominates for butirosin while the 5"-OH is favored for neomycin. These results open the potential for the rational design of aminoglycoside kinase inhibitors based on functionalization of either the 6-aminohexose or the pentose rings of aminoglycoside antibiotics.

Aminoglycosides↗

PubMed1997

Biologically important conformations of aminoglycoside antibiotics bound to an aminoglycoside 3'-phosphotransferase as determined by transferred nuclear Overhauser effect spectroscopy.

NMR spectroscopy has been used to study the interaction of aminoglycoside antibiotics with an aminoglycoside antibiotic 3'-phosphotransferase [APH(3')-IIIa]. APH(3')-IIIa is an enterococcal enzyme that is responsible for the ATP-dependent O-phosphorylation of a broad range of aminoglycoside antibiotics. The NMR method of transferred nuclear Overhauser effect spectroscopy (TRNOESY) was used to detect intra- and inter-ring NOEs for butirosin A and amikacin in their respective ternary complexes with APH(3')-IIIa and ATP. NOE-derived distance constraints were used in energy minimization and dynamics routines to yield enzyme-bound structures for butirosin A. These structures suggest that the 2,6-diamino-2,6-dideoxy-D-glucose and D-xylose rings have restricted motions and are in a stacking arrangement. The TRNOE spectra for amikacin suggest that the 6-amino-6-deoxy-D-glucose ring is flexible when the antibiotic is bound to APH(3')-IIIa. The 15N resonances of butirosin A were assigned and the pKa values of the amino groups of butirosin A and amikacin were determined by 15N NMR spectroscopy. The N3 amino groups of butirosin A and amikacin have lowered pKa values, which is attributed to the (S)-4-amino-2-hydroxybutyryl (AHB) group of the antibiotics. This work provides an insight into the geometrical and electrostatic nature of aminoglycoside antibiotics bound to a modifying enzyme and will provide a basis for the design of inhibitors of APH(3')-IIIa.

Amikacin↗

PubMed1997

Expression, purification, and characterization of enzyme IIA(glc) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum.

The gene encoding enzyme IIA(glc) (EIIA) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum was cloned into a regulated expression vector. The purified protein product of the overexpressed gene was characterized as an active phosphoacceptor from HPr with a higher pI than previously described EIIAs. M. capricolum EIIA was unreactive with antibodies directed against the corresponding proteins from either Gram-positive or Gram-negative bacteria. Enzyme IIA(glc) behaved as a homogeneous, monomeric species of 16,700 Mr in analytical ultracentrifugation. The circular dichroism far-UV spectrum of EIIA reflects a low alpha-helical content and predominantly beta-sheet structural content: temperature-induced changes in ellipticity at 205 nm showed that the protein undergoes reversible, two-state thermal unfolding with Tm = 70.0 +/- 0.3 degrees C and a van't Hoff deltaH of 90 kcal/mol. Enzyme I (64,600 Mr) from M. capricolum exhibited a monomer-dimer-tetramer association at 4 and 20 degrees C with dimerization constants of log K(A) = 5.6 and 5.1 [M(-1)], respectively, in sedimentation equilibrium experiments. A new vector, capable of introducing an N-terminal His tag on a protein, was developed in order to generate highly purified heat-stable protein (HPr). No significant interaction of EIIA with HPr was detected by gel-filtration chromatography, intrinsic tryptophanyl residue fluorescence changes, titration calorimetry, biomolecular interaction, or sedimentation equilibrium studies. While Escherichia coli EIIA inhibits Gram-negative glycerol kinase activity, the M. capricolum EIIA has no effect on the homologous glycerol kinase. The probable regulator of sugar transport systems, HPr(Ser) kinase, was demonstrated in extracts of M. capricolum and Mycoplasma genitalium. Gene mapping studies demonstrated that, in contrast to the clustered arrangement of genes encoding HPr and enzyme I in E. coli, these genes are located diametrically opposite in the M. capricolum chromosome.

Chromosome Mapping↗

PubMed2000

Topography of the surface of the Escherichia coli phosphotransferase system protein enzyme IIAglc that interacts with lactose permease.

The unphosphorylated form of enzyme IIAglc of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system inhibits transport catalyzed by lactose permease. We (Seok et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 13515-13519) previously characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIAglc, using radioactive enzyme IIAglc. Subsequent studies (Sondej et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530) suggested consensus binding sequences on proteins that interact with enzyme IIAglc. The present study characterizes a region on the surface of enzyme IIAglc that interfaces with lactose permease. Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIAglc, but not lactose permease, reduced the degree of interaction between the two proteins. To localize the lysine residue(s) on enzyme IIAglc that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIAglc in a test for binding to lactose permease. The Lys69 mutant was also defective in phosphoryl transfer to glucose permease. To derive further information concerning the contact surface, additional selected residues in the vicinity of Lys69 were mutagenized and tested for binding to lactose permease. On the basis of these studies, a model for the region of the surface of enzyme IIAglc that interacts with lactose permease is proposed.

Acetates↗

PubMed2000

The glucose transporter of the Escherichia coli phosphotransferase system: linker insertion mutants and split variants.

The IICB(Glc) subunit of the glucose transporter acts by a mechanism which couples vectorial translocation with phosphorylation of the substrate. It contains 8 transmembrane segments connected by 4 periplasmic, 2 short, 1 long (80 residues), cytoplasmic loops and an independently folding cytoplasmic domain at the C-terminus. Random DNase I cleavage, EcoRI linker insertion, and screening for transport-active mutants afforded 12 variants with between 46% and 116% of wild-type sugar phosphorylation activity. They carried inserts of up to 29 residues and short deletions in periplasmic loops 1, 2, and 3, in the long cytoplasmic loop 3, and in the linker region between the membrane spanning IIC(Glc) and the cytoplasmic IIB(Glc) domains. Disruption of the gene at the sites of linker insertion decreased the expression level and diminished phosphotransferase activity to between 7% and 32%. IICB(Glc) with a discontinuity in the cytoplasmic loop was purified to homogeneity as a stable complex. It was active only if encoded by a dicistronic operon but not if encoded by two genes on two different replicons, suggesting that spatial proximity of the nascent polypeptide chains is important for folding and membrane assembly.

Amino Acid Sequence↗

PubMed2001

NMR-restrained docking of a peptidic inhibitor to the N-terminal domain of the phosphoenolpyruvate:sugar phosphotransferase enzyme I.

Starting from the NMR structure of the binary complex between the N-terminal domain of the unphosphorylated enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase (PTS) and the histidine-containing phosphocarrier protein (HPr), a molecular model of the phosphorylated transition state of the related complex was established using constrained simulated annealing. The coordinates of the phosphorylated EIN enzyme were then used in a second step for flexible docking of a decapeptide inhibitor of EIN whose enzyme-bound conformation itself was determined by NMR using transferred nuclear Overhauser effects. Two phosphorylation models of the peptide inhibitor were investigated and shown to be both functional. Interestingly, one model is very similar to that of the complex between EIN and its natural substrate HPr. The present study demonstrates that NMR-guided flexible docking constitutes an interesting tool for docking highly flexible peptide ligands and facilitates the upcoming protein-based design of nonpeptide EIN inhibitors for discovering new antibiotics.

Amino Acid Sequence↗

PubMed2004

Cloning, sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system, glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998.

A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109. Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E. coli (Gene Bank accession no. J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no. Y14603). The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da. The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos. J02708, AE016987 and D90892 respectively). The protein sequence showed significant homology to that of the phosphotransferase system i.e. the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522). The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination. Mutants obtained were unable to grow on minimal medium with sorbitol. The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation.

Amino Acid Sequence↗

PubMed1990

Targeted insertion of the neomycin phosphotransferase gene into the tubulin gene cluster of Trypanosoma brucei.

Kinetoplastids are unicellular eukaryotes that include important parasites of man, such as trypanosomes and leishmanias. The study of these organisms received a recent boost from the development of transient transformation allowing the short-term expression of genes reintroduced into parasites like Trypanosoma brucei, the causative agent of African trypanosomiasis. We have obtained long-term stable transformants of T. brucei that have acquired the ability to grow in medium containing the drug G418, following the targeted insertion of the bacterial gene for neomycin phosphotransferase (neo(r) gene) into the trypanosome tubulin cluster. Plasmids in which part of the T. brucei tubulin gene cluster has been replaced by the neo(r) gene were used. Targeting efficiency was higher with a linearized than with a circular construct, and with 5 kilobases of tubulin gene cluster than with 0.9 kilobases. With these neo(r) constructs homologous recombination seems to be the preferred route for insertion of exogenous DNA into the trypanosome genome, allowing gene targeting without counter-selection.

Animals↗

PubMed1993

Purification and characterization of microbially expressed neomycin phosphotransferase II (NPTII) protein and its equivalence to the plant expressed protein.

The gene encoding neomycin phosphotransferase II (NPTII) has been used routinely as a selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the same coding sequence used for plant transformation was introduced into Escherichia coli to produce gram quantities of this protein. A unique, simple, rapid and efficient purification method was developed to purify thirty grams of NPTII protein. The microbially produced NPTII was shown to be chemically and functionally equivalent to the NPTII protein expressed in and purified from genetically engineered cotton seed, potato tubers and tomato fruit. Microbially produced and plant produced NPTII proteins have comparable molecular weights, immuno-reactivities, epitope structures, amino terminal amino acid sequences, biological activities and both lack glycosylation. Demonstrating the equivalence of NPTII protein from these sources establishes the validity of using the microbially produced NPTII to assess the safety of the NPTII protein produced in genetically engineered crops.

Amino Acid Sequence↗

PubMed1970

The role of the phosphoenolpyruvate phosphotransferase system in the transport of N-acetyl-D-glucosamine by Escherichia coli.

The properties of an N-acetyl-d-glucosamine-transport system have been studied by following the intracellular accumulation of methyl 2-acetamido-2-deoxy-alpha-d-[1-(14)C]glucoside by Escherichia coli. The same analogue was used to assay phosphoenolpyruvate phosphotransferase activity of toluene-treated cells. Transport and phosphorylation are induced by growth on d-glucosamine or N-acetyl-d-glucosamine. Mutants resistant to N-iodoacetyl-d-glucosamine are defective in the uptake and phosphorylation of the labelled glycoside.

Biological Transport, Active↗

PubMed1985

Pyrophosphate:fructose 6-phosphate 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher plants.

The activity of pyrophosphate:fructose-6-phosphate 1-phosphotransferase [PFK (PPi); EC 2.7.1.90] in extracts of the storage tissues of leek (Allium porrum), beetroot (Beta vulgaris) and roots of darnel (Lolium temulentum) exceeded 0.15 mumol/min per g fresh wt. As net flux from fructose 1,6-bisphosphate to fructose 6-phosphate in these tissues is unlikely, it is suggested that PFK (PPi) does not contribute to gluconeogenesis or starch synthesis. The maximum catalytic activities of PFK (PPi) in apex, stele and cortex of the root of pea (Pisum sativum) and in the developing and the thermogenic club of the spadix of cuckoo-pint (Arum maculatum) were measured and compared with those of phosphofructokinase, and to estimates of the rates of carbohydrate oxidation. PPi and fructose 2,6-bisphosphate in Arum clubs were measured. The above measurements are consistent with a glycolytic role for PFK (PPi) in tissues where there is marked biosynthesis, but not in the thermogenic club of Arum. The possibility that PFK (PPi) is a means of synthesizing pyrophosphate is discussed.

Fructosediphosphates↗

PubMed1987

Altered molecular size of N-acetylglucosamine 1-phosphotransferase in I-cell disease and pseudo-Hurler polydystrophy.

The size of the mutant N-acetylglucosamine 1-phosphotransferase in Golgi membranes from fibroblasts of patients with I-cell disease and classical pseudo-Hurler polydystrophy, which comprised one complementation group characterized by deficiency towards both artificial and natural acceptor substrates, was significantly smaller than the normal enzyme, 151-174 kDa compared with 225-278 kDa. The size of the mutant enzyme from cell lines of patients with variant forms of pseudo-Hurler polydystrophy, which comprised another complementation group characterized by normal activity towards mono- and oligo-saccharide substrates, was significantly larger than the normal enzyme, ranging from 321 to 356 kDa in two families and from 528 to 547 kDa in a third family. These findings suggest that the mutations in I-cell disease and classical pseudo-Hurler polydystrophy result in a missing enzyme component, which renders the enzyme catalytically inefficient toward any type of acceptor substrate. In contrast, the mutations in the variant forms of pseudo-Hurler polydystrophy produce a larger enzyme molecule which is active toward small substrates but is incapable of binding natural lysosomal glycoprotein substrates.

Cell Line↗