SEARCH · PubMed Health

Results for “MANNITOL”

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 19 recordsLinked to original sources

PubMed1971

Catabolism of fructose and mannitol in Clostridium thermocellum: presence of phosphoenolpyruvate: fructose phosphotransferase, fructose 1-phosphate kinase, phosphoenolpyruvate: mannitol phosphotransferase, and mannitol 1-phosphate dehydrogenase in cell extracts.

Fructose and mannitol are fermented by Clostridium thermocellum in a medium containing salts and 0.5% yeast extract. The initial reaction in the catabolism of fructose was found to be the formation of fructose l-phosphate by phosphoenolpyruvate (PEP):fructose phosphotransferase which resembles the Kundig-Roseman phosphotransferase system. The phosphorylation of fructose l-phosphate to form fructose-1, 6-diphosphate is catalyzed by fructose l-phosphate kinase. Fructose-1, 6-diphosphate can be further metabolized by the Embden-Meyerhof pathway. The formation of both PEP:fructose phosphotransferase and fructose l-phosphate kinase is induced by growth in fructose medium. Mannitol catabolism was found to proceed by the phosphorylation of mannitol by PEP:mannitol phosphotransferase to form mannitol l-phosphate. Mannitol l-phosphate is converted to fructose 6-phosphate by a nicotinamide adenine dinucleotide-specific mannitol l-phosphate dehydrogenase. The fructose 6-phosphate formed in the reaction can enter the glycolytic scheme. The formation of both PEP:mannitol phosphotransferase and mannitol l-phosphate dehydrogenase is induced by growth in mannitol medium. Evidence is presented for the induction by mannitol of PEP:mannitol phosphotransferase and mannitol l-phosphate dehydrogenase in suspensions of fructose-grown cells.

Alcohol Oxidoreductases↗

PubMed1994

Expression, purification, and kinetic characterization of the mannitol transport domain of the phosphoenolpyruvate-dependent mannitol phosphotransferase system of Escherichia coli. Kinetic evidence that the E. coli mannitol transport protein is a functional dimer.

The overexpression of the membrane-bound C domain of the mannitol transport protein EIIMtl of Escherichia coli has been achieved. This protein, IICMtl, consisting of the first 346 amino acids, was purified from membrane vesicles and still bound mannitol with a high affinity. Gel filtration experiments showed that purified IICMtl was a dimer, confirming that the interaction within the EIIMtl dimer occurs between the membrane-bound portions of the protein. IICMtl in combination with a chimeric protein consisting of the membrane-bound EIIGlc C domain and the cytoplasmic EIIMtl BA domain could restore both phosphoenolpyruvate-dependent phosphorylation and mannitol/mannitol-P exchange activity. The interaction in this complex was comparable to that of IICMtl with soluble IIBAMtl in as much as there appeared to be no specific interaction between IICMtl and the membrane-bound EIIGlc C domain; the Km of IICMtl for the chimer was so low that saturation could not be achieved. In contrast, a very high affinity with a Km of 2 nM was measured between purified IICMtl and purified EIIMtl. This interaction was manifested in a IICMtl-dependent stimulation of the EIIMtl catalyzed phosphoenolpyruvate-dependent mannitol phosphorylation reaction and the mannitol/mannitol-P exchange reaction. The high affinity of IICMtl for the wild type enzyme can be explained by the formation of heterodimers consisting of a IICMtl monomer and an EIIMtl monomer which interact at the level of the membrane-bound domains. The 2-fold increase in mannitol phosphorylation activity of the hetero- versus homodimer is an indication that the individual subunits in the homodimer are functionally coupled and work at only half their maximum rate. It is known that the EIIMtl dimer, but not the monomer, catalyzes the mannitol/mannitol-P exchange reaction. Since the heterodimer also catalyzes this reaction, it appears that only one functional B domain is required per dimer.

Base Sequence↗

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↗

PubMed2000

Mannitol for acute traumatic brain injury.

BACKGROUND: Mannitol is sometimes dramatically effective in reversing acute brain swelling, but its effectiveness in the on-going management of severe head injury remains open to question. There is evidence that in prolonged dosage mannitol may pass from the blood into the brain, where it might cause reverse osmotic shifts that increase intracranial pressure. OBJECTIVES: To assess the effects of different mannitol therapy regimens, of mannitol compared to other intracranial pressure (ICP) lowering agents, and to quantify the effectiveness of mannitol administration given at other stages following acute traumatic brain injury. SEARCH STRATEGY: The review drew on the search strategy for the Injuries Group as a whole. We checked reference lists of trials and review articles, and contacted authors of trials. SELECTION CRITERIA: Randomised trials of mannitol, in patients with acute traumatic brain injury of any severity. The comparison group could be placebo-controlled, no drug, different dose, or different drug. Trials where the intervention was started more than eight weeks after injury, and cross-over trials were excluded. DATA COLLECTION AND ANALYSIS: The reviewers independently rated quality of allocation concealment and extracted the data. Relative risks (RR) and 95% confidence intervals (CI) were calculated for each trial on an intention to treat basis. MAIN RESULTS: Overall there were few eligible trials. There were no trials comparing different doses, or type of administration. One trial compared ICP-directed therapy to 'standard care' (RR for death= 0.83; 95% CI 0.47;1.46). One trial compared mannitol to pentobarbital (RR for death = 0.85; 95% CI 0. 52;1.38). No trials compared mannitol to other ICP lowering agents. One trial tested the effectiveness of pre-hospital administration of mannitol against placebo (RR for death=1.59; 95% CI 0.44;5.79). REVIEWER'S CONCLUSIONS: There are insufficient data to recommend one form of mannitol infusion over another. Mannitol therapy for raised ICP may have a beneficial effect on mortality when compared to pentobarbital treatment. ICP-directed treatment shows a small beneficial effect compared to treatment directed by neurological signs and physiological indicators. There are insufficient data on the effectiveness of pre-hospital administration of mannitol to preclude either a harmful or a beneficial effect on mortality.

Brain Injuries↗

PubMed1998

The physical state of mannitol after freeze-drying: effects of mannitol concentration, freezing rate, and a noncrystallizing cosolute.

The objectives of this study were to (1) measure the effects of freezing rate and mannitol concentration on the physical state of freeze-dried mannitol when mannitol is present as a single component, (2) determine the relative concentration threshold above which crystalline mannitol can be observed by X-ray powder diffraction in the freeze-dried solid when a variety of noncrystallizing solutes are included in the formulation, and (3) measure the glass transition temperature of amorphous mannitol and to determine the degree to which the glass transition temperature of freeze-dried solids consisting of mannitol and a disaccharide is predicted by the Gordon-Taylor equation. Both freezing rate and mannitol concentration influence the crystal form of mannitol in the freeze-dried solid when mannitol is present as a single component. Slow freezing of 10% (w/v) mannitol produces a mixture of the alpha and beta polymorphs, whereas fast freezing of the same solution produces the delta form. Fast freezing of 5% (w/v) mannitol results primarily in the beta form. The threshold concentration above which crystalline mannitol is detected in the freeze-dried solid by X-ray diffraction is consistently about 30% (w/w) when a second, noncrystallizing solute is present, regardless of the nature of the second component. The glass transition temperature of amorphous mannitol measured from the quench-cooled melt is approximately 13 degreesC. Accordingly, mannitol is an effective plasticizer of freeze-dried solids when the mannitol remains amorphous. Glass transition temperatures of mixtures of mannitol and the disaccharides sucrose, maltose, trehalose, and lactose are well predicted by the Gordon-Taylor equation with values of k in the range of 3 to 4.

Chemical Phenomena↗

PubMed2004

Effect of vacuum drying on protein-mannitol interactions: the physical state of mannitol and protein structure in the dried state.

The purpose of the present studies was to systematically investigate protein-mannitol interactions using vacuum drying, to obtain a better understanding of the effect of protein/mannitol wt/wt ratios on the physical state of mannitol and protein secondary structure in the dried state. Solutions containing beta-lactoglobulin (betaLg):mannitol (1:1-1:15 wt/wt) were vacuum dried at 5 degrees C under 3000 mTorr of pressure. The physical state of mannitol was studied using x-ray powder diffractometry (XRPD), polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and modulated differential scanning calorimetry (MDSC). XRPD studies indicated that mannitol remained amorphous up to 1:5 wt/wt betaLg:mannitol ratio, whereas PLM showed the presence of crystals of mannitol in all dried samples except for the 1:1 wt/wt betaLg:mannitol dried sample. FTIR studies indicated that a small proportion of crystalline mannitol was present along with the amorphous mannitol in dried samples at lower (less than 1:5 wt/wt) betaLg:mannitol ratios. The T(g) of the dried 1:1 wt/wt betaLg:mannitol sample was observed at 33.4 degrees C in MDSC studies, which indicated that at least a part of mannitol co-existed with protein in a single amorphous phase. Evaluation of the crystallization exotherms indicated that irrespective of the betaLg:protein wt/wt ratio in the initial sample, the protein to amorphous mannitol ratio was below 1:1 wt/wt in all dried samples. Second-derivative FTIR studies on dried betaLg and recombinant human interferon alpha-2a samples showed that mannitol affected protein secondary structure to a varying degree depending on the overall mannitol content in the dried sample and the type of protein.

Calorimetry, Differential Scanning↗

PubMed2004

Engineering Lactococcus lactis for production of mannitol: high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system.

Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTS(Mtl)). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (Delta ldh Delta mtlA and Delta ldh Delta mtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo (13)C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTS(Mtl). Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.

Base Sequence↗

PubMed2005

Overproduction of heterologous mannitol 1-phosphatase: a key factor for engineering mannitol production by Lactococcus lactis.

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.

Animals↗

PubMed1991

Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes.

Enzyme IIIMtl is part of the mannitol phosphotransferase system of Enterococcus faecalis. It is phosphorylated in a reaction sequence requiring enzyme I and heat-stable phosphocarrier protein (HPr). The phospho group is transferred from enzyme IIIMtl to enzyme IIMtl, which then catalyzes the uptake and concomitant phosphorylation of mannitol. The internalized mannitol-1-phosphate is oxidized to fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. In this report we describe the cloning of the mtlF and mtlD genes, encoding enzyme IIIMtl and mannitol-1-phosphate dehydrogenase of E. faecalis, by a complementation system designed for cloning of gram-positive phosphotransferase system genes. The complete nucleotide sequences of mtlF, mtlD, and flanking regions were determined. From the gene sequences, the primary translation products are deduced to consist of 145 amino acids (enzyme IIIMtl) and 374 amino acids (mannitol-1-phosphate dehydrogenase). Amino acid sequence comparison confirmed a 41% similarity of E. faecalis enzyme IIIMtl to the hydrophilic enzyme IIIMtl-like portion of enzyme IIMtl of Escherichia coli and 45% similarity to enzyme IIIMtl of Staphylococcus carnosus. The putative N-terminal NAD+ binding domain of mannitol-1-phosphate dehydrogenase of E. faecalis shows a high degree of similarity with the N terminus of E. coli mannitol-1-phosphate dehydrogenase (T. Davis, M. Yamada, M. Elgort, and M. H. Saier, Jr., Mol. Microbiol. 2:405-412, 1988) and the N-terminal part of the translation product of S. carnosus mtlD, which was also determined in this study. There is 40% similarity between the dehydrogenases of E. faecalis and E. coli over the whole length of the enzymes. The organization of mannitol-specific genes in E. faecalis seems to be similar to the organization in S. carnosus. The open reading frame for enzyme IIIMtl E. faecalis is followed by a stem-loop structure, analogous to a typical Rho-independent terminator. We conclude that the mannitol-specific genes are organized in an operon and that the gene order is mtlA orfX mtlF mtlD.

Amino Acid Sequence↗

PubMed2002

Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata.

Our previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen-mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non-mannitol-containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol- and non-mannitol-secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non-mannitol-secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol-secreting fungal plant pathogens.

Alternaria↗

PubMed1965

MANNITOL AND MANNITOL DEHYDROGENASES IN CONIDIA OF ASPERGILLUS ORYZAE.

Horikoshi, Koki (The Institute of Physical and Chemical Research, Tokyo, Japan), Shigeji Iida, and Yonosuke Ikeda. Mannitol and mannitol dehydrogenases in conidia of Aspergillus oryzae. J. Bacteriol. 89:326-330. 1965.-A sugar alcohol was isolated from the conidia of Aspergillus oryzae and identified as d-mannitol. Two types of d-mannitol dehydrogenases, nicotinamide adenine dinucleotide phosphate-linked and nicotinamide adenine dinucleotide-linked, were found in the conidia. Substrate specificities, pH optima, Michaelis-Menton constants, and the effects of inhibitors were studied. d-Mannitol was converted to fructose by the dehydrogenases. Synthesis of d-mannitol dehydrogenases was not observed during germination; the content of d-mannitol decreased at an early stage of germination. It was assumed, therefore, that d-mannitol might be used as the source of endogenous respiration and provide energy for the germination.

Aspergillus↗

PubMed1989

Deletion mutants of the Escherichia coli K-12 mannitol permease: dissection of transport-phosphorylation, phospho-exchange, and mannitol-binding activities.

We have constructed a series of deletion mutations of the cloned Escherichia coli K-12 mtlA gene, which encodes the mannitol-specific enzyme II of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system. This membrane-bound permease consists of 637 amino acid residues and is responsible for the concomitant transport and phosphorylation of D-mannitol in E. coli. Deletions into the 3' end of mtlA were constructed by exonuclease III digestion. Restriction mapping of the resultant plasmids identified several classes of deletions that lacked approximately 5% to more than 75% of the gene. Immunoblotting experiments revealed that many of these plasmids expressed proteins within the size range predicted by the restriction analyses, and all of these proteins were membrane localized, which demonstrated that none of the C-terminal half of the permease is required for membrane insertion. Functional analyses of the deletion proteins, expressed in an E. coli strain deleted for the chromosomal copy of mtlA, showed that all but one of the strains containing confirmed deletions were inactive in transport and PEP-dependent phosphorylation of mannitol, but deletions removing up to at least 117 amino acid residues from the C terminus of the permease were still active in catalyzing phospho exchange between mannitol 1-phosphate and mannitol. A deletion protein that lacked 240 residues from the C terminus of the permease was inactive in phospho exchange but still bound mannitol with high affinity. These experiments localize sites important for transport and PEP-dependent phosphorylation to the extreme C terminus of the mannitol permease, sites important for phospho exchange to between residues 377 and 519, and sites necessary for mannitol binding to the N-terminal 60% of the molecule. The results are discussed with respect to the fact that the mannitol permease consists of structurally independent N- and C-terminal domains.

Binding Sites↗

PubMed1998

Mannitol 1-phosphate mediates an inhibitory effect of mannitol on the activity and the translocation of glucokinase in isolated rat hepatocytes.

When tested in the presence of an inhibitor of sorbitol dehydrogenase, both mannitol and sorbitol caused a progressive inhibition of the detritiation of [2-3H]glucose in isolated rat hepatocytes. The purpose of the present work was to investigate the possibility that this effect was mediated by the regulatory protein of glucokinase. When added to hepatocytes, mannitol decreased the apparent affinity of glucokinase for glucose and increased the concentration of fructose required to stimulate detritiation, without affecting the concentration of fructose 1-phosphate. Its effect could be attributed to the formation of mannitol 1-phosphate, a potent agonist of the regulatory protein, which, similarly to fructose 6-phosphate, reinforces its inhibitory action. Formation of mannitol 1-phosphate in hepatocytes was dependent on the presence of mannitol and was stimulated by compounds that increase the concentration of glucose 6-phosphate. Liver extracts catalysed the conversion of mannitol to mannitol 1-phosphate about 7 times more rapidly in the presence of glucose 6-phosphate than of ATP. The glucose 6-phosphate-dependent formation was entirely accounted for by a microsomal enzyme, glucose-6-phosphatase and was not due to a loss of latency of this enzyme. In hepatocytes in primary culture, mannitol decreased the detritiation rate and counteracted the effect of fructose to stimulate glucokinase translocation. Taken together, these results strongly support a central role played by the regulatory protein in the control of glucokinase activity and translocation in the liver, as well as a feedback control exerted by fructose 6-phosphate on this enzyme.

Animals↗

PubMed1981

Improved enzymatic method for determining mannitol and its application to dog serum after mannitol infusion.

An enzymatic method for mannitol quantitation has been developed. Its application mannitol levels in serum samples from dogs after mannitol infusion is illustrated. The method is based on the spectrophotometric measurement of the initial rate of NADH formation in the reaction between mannitol and NAD catalyzed by mannitol dehydrogenase. A linear relationship between initial rates and mannitol concentration is seen between 17 mumol/l and 6.7 mmol/l mannitol. From the extent of dilution of the serum sample in the assay this corresponds to serum levels of 0.5 to 200 mmol/l. Because of the high degree of substrate specificity of mannitol dehydrogenase and the extensive dilution of the serum sample in the assay reaction mixture, serum proteins and glucose do not interfere with the reaction. As a result, pre-treatment of samples to remove glucose and deproteinization are necessary.

Animals↗

PubMed1992

Liquid volume, Na+ and mannitol concentration in a hypertonic mannitol-Ringer hydrothorax.

In anesthetised rabbits with a 2 ml hypertonic mannitol-Ringer hydrothorax in the right space 30 mM/L mannitol were required for an unchanged volume of the hydrothorax after 60 min. [Na+] in the pleural liquid 10, 30 and 60 min after this hydrothorax was 8, 7 and 5 mEq/L, respectively, lower (P less than 0.01) than the initial one and that in a Ringer-hydrothorax. This seems due to the active transport of Na+ out of the pleural space followed by little water because of the osmotic pressure exerted by mannitol. This finding provides further evidence for an active transport without using inhibitors, and implies that the mesothelium offers an appreciable resistance to small solute diffusion. Mannitol concentration, measured at corresponding times from the activity of labeled mannitol, was 76, 68 and 56%, respectively, of the initial one (24.5 mM/L). From 30 to 60 min 6.5 microM of mannitol left the right space mainly by diffusion. The diffusional permeability of the mesothelium was indirectly assessed from the diffusional outflux of mannitol, the surface of the pleural space, and an estimate of mannitol concentration in the interstitium next to the mesothelium: it is smaller than that found in vitro.

Animals↗

PubMed2004

Possible roles for mannitol and mannitol dehydrogenase in the biotrophic plant pathogen Uromyces fabae.

Levels of the C6-polyol mannitol were observed to rise dramatically in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant Vicia faba. Mannitol was found in millimolar concentrations in extracts and apoplastic fluids of infected leaves and also in extracts of spores. We suggest that this polyol might have at least a dual function: first, as a carbohydrate storage compound, and second, as a scavenger of reactive oxygen species. Mannitol accumulation is accompanied by high expression of a mannitol dehydrogenase (MAD1) in haustoria. While MAD1 transcripts were detected in haustoria only, immunolocalization studies show that the gene product is also present in spores. Kinetic and thermodynamic analyses of the MAD1p catalyzed reactions indicate that the enzyme might be responsible for the production of mannitol in haustoria and for the utilization of mannitol in spores. Since V. faba is normally unable to synthesize or utilize polyols, the multipurpose usage of mannitol seems an ideal strategy for the fungal pathogen.

Basidiomycota↗

PubMed1995

Phosphorylation site mutants of the mannitol transport protein enzyme IImtl of Escherichia coli: studies on the interaction between the mannitol translocating C-domain and the phosphorylation site on the energy-coupling B-domain.

Mannitol binding and translocation catalyzed by the C domain of the Escherichia coli mannitol transport protein enzyme IImtl is influenced by domain B. This interaction was studied by monitoring the effects of mutating the B domain phosphorylation site, C384, on the kinetics of mannitol binding to the C domain. The dissociation constants for mannitol to the C384 mutants in inside-out membrane vesicles varied from 45 nM for the wild-type enzyme to 306 nM for the mutants. The rate constants pertinent to the binding equilibrium were also altered by the mutations. The association rate of mannitol to the cytoplasmic binding site in the mutants was accelerated for all mutants. The exchange rate of bound mannitol on the wild-type enzyme was shown to be pH dependent with a pKa of approximately 8 and increasing rates at higher pH. This rate was increased for all the mutants, but the pKas differed for the various mutants. The exchange rate for binding to the isolated IICmtl, however, was not pH dependent and exhibited a low rate. Exchange measured at 4 degrees C showed that, of the two steps, binding and occlusion, involved in binding to wild-type EIImtl in inside-out vesicles, only one could be detected for the C384E and C384L mutants. This suggests that the mutations increased the rate of the occlusion step so that it was no longer separable from the initial binding step or that the mutations eliminated the occlusion step altogether. The change in the mannitol binding kinetics of the C domain indicates that the B and C domains of EIImtl influence each other's conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1996

Relation between the oligomerization state and the transport and phosphorylation function of the Escherichia coli mannitol transport protein: interaction between mannitol-specific enzyme II monomers studied by complementation of inactive site-directed mutants.

Previous experiments with the mannitol-specific enzyme II of Escherichia coli, EIImtl, have demonstrated that (1) the enzyme is a dimer, (2) the dimer is necessary for maximum activity, and (3) phosphoryl groups could be transferred between EIImtl subunits [van Weeghel et al. (1991) Biochemistry 30, 1768-1773; Weng et al. (1992) J. Biol. Chem. 267, 19529-19535; Weng & Jacobson (1993) Biochemistry 32, 11211-11216; Stolz et al. (1993) J. Biol. Chem. 268, 27094-27099]. The experiments in this article address the mechanistic role of the dimer. They indicate that the A, B, and C domains of EIImtl preferentially interact within the same subunit. Site-directed mutants in each of the three domains of EIImtl were used to study phosphoryl group transfer by the EIImtl dimer in vitro and mannitol transport in vivo. The C domain mutant, EIImtl-G196D, which was unable to bind mannitol, and the separated C domain, IICmtl, which was unable to phosphorylate mannitol, formed a heterodimer which was capable of mannitol phosphorylation in vitro and mannitol transport in vivo. The rates of phosphorylation were approximately 10-fold lower in heterodimers containing two inactive subunits relative to the rates in heterodimers containing one inactive and one wild type subunit; phosphoryl group transfer through one subunit is kinetically preferred to intersubunit transfer. Heterodimers formed in vivo between one wild type EIImtl subunit and the CB domain double mutant, EIImtl-G196D/C384S, transported mannitol as rapidly as wild type EIImtl alone; the presence of the inactive double mutant subunit did not reduce the transport rate. Thus, only one active A, B, and C domain in the dimer is sufficient for transport and phosphorylation activity, and if all three domains are situated on the same subunit, maximum rates are achieved.

Biological Transport↗