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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↗
PubMed2003

Mannitol-1-phosphate dehydrogenase (MtlD) is required for mannitol and glucitol assimilation in Bacillus subtilis: possible cooperation of mtl and gut operons.

We found that mannitol-1-phosphate dehydrogenase (MtlD), a component of the mannitol-specific phosphotransferase system, is required for glucitol assimilation in addition to GutR, GutB, and GutP in Bacillus subtilis. Northern hybridization of total RNA and microarray studies of RNA from cells cultured on glucose, mannitol, and glucitol indicated that mannitol as the sole carbon source induced hyperexpression of the mtl operon, whereas glucitol induced both mtl and gut operons. The B. subtilis mtl operon consists of mtlA (encoding enzyme IICBA(mt1)) and mtlD, and its transcriptional regulator gene, mtlR, is located 14.4 kb downstream from the mtl operon on the chromosome. The mtlA, mtlD, and mtlR mutants disrupted by the introduction of the pMUTin derivatives MTLAd, MTLDd, and MTLRd, respectively, could not grow normally on either mannitol or glucitol. However, the growth of MTLAd on glucitol was enhanced by IPTG (isopropyl-beta-D-thiogalactopyranoside). This mutant has an IPTG-inducible promoter (Pspac promoter) located in mtlA, and this site corresponds to the upstream region of mtlD. Insertion mutants of mtlD harboring the chloramphenicol resistance gene also could not grow on either mannitol or glucitol. In contrast, an insertion mutant of mtlA could grow on glucitol but not on mannitol in the presence or absence of IPTG. MtlR bound to the promoter region of the mtl operon but not to a DNA fragment containing the gut promoter region.

Bacillus subtilis↗
PubMed2001

Nitrophenide (Megasul) blocks Eimeria tenella development by inhibiting the mannitol cycle enzyme mannitol-1-phosphate dehydrogenase.

Unsporulated oocysts of the protozoan parasite Eimeria tenella contain high levels of mannitol, which is thought to be the principal energy source for the process of sporulation. Biosynthesis and utilization of this sugar alcohol occurs via a metabolic pathway known as the mannitol cycle. Here, results are presented that suggest that 3-nitrophenyl disulfide (nitrophenide, Megasul), an anticoccidial drug commercially used in the 1950s, inhibits mannitol-1-phosphate dehydrogenase (M1PDH), which catalyzes the committed enzymatic step in the mannitol cycle. Treatment of E. tenella-infected chickens with nitrophenide resulted in a 90% reduction in oocyst shedding. The remaining oocysts displayed significant morphological abnormalities and were largely incapable of further development. Nitrophenide treatment did not affect parasite asexual reproduction, suggesting specificity for the sexual stage of the life cycle. Isolated oocysts from chickens treated with nitrophenide exhibited a dose-dependent reduction in mannitol, suggesting in vivo inhibition of parasite mannitol biosynthesis. Nitrophenide-mediated inhibition of MIPDH was observed in vitro using purified native enzyme. Moreover, MIPDH activity immunoprecipitated from E. tenella-infected cecal tissues was significantly lower in nitrophenide-treated compared with untreated chickens. Western blot analysis and immunohistochemistry showed that parasites from nitrophenide-treated and untreated chickens contained similar enzyme levels. These data suggest that nitrophenide blocks parasite development at the sexual stages by targeting M1PDH. Thus, targeting of the mannitol cycle with drugs could provide an avenue for controlling the spread of E. tenella in commercial production facilities by preventing oocyst shedding.

Animals↗
PubMed1986

Mannitol concentrations in blood plasma in connection with transurethral resection of the prostate using mannitol solution as an irrigating fluid.

Thirteen patients undergoing transurethral resections of the prostate (TURP) using iso-osmolar 5% mannitol as an irrigating fluid were studied. Mannitol was determined in serum (plasma), as were sodium, prostatic acid phosphatase protein (PAP) and osmolality as probable indicators of absorption of irrigating fluid. The plasma level of mannitol (mean 2.7 g/l = 15 mmol/l) immediately postoperatively, the increase in serum PAP (mean 93 micrograms/l) and the decrease in serum sodium (mean 8.7 mmol/l) all reflect the amount of irrigating fluid absorbed during TURP. The three variables are intercorrelated. The plasma osmolality was unchanged (mean -1 mosmol/kg). A small but constant fraction of mannitol was found in the erythrocytes 2 hours after the operation, amounting to about 3% of the simultaneous plasma concentration. The mean plasma half-life of mannitol was 127 min in the absence of uraemia. In two cases showing a slight increase in serum creatinine the half-lives were prolonged. An estimate of the volume of fluid absorbed was made from the observed plasma mannitol levels. A fluid absorption of up to 3 litres (mean 1.1 l) was found. A strong diuretic effect was observed in some cases when irrigation with mannitol was combined with i.v. furosemide. We conclude that the i.v. diuretic should be withheld until the extent of fluid absorption has been estimated. If the sodium concentration in the serum is largely unchanged immediately postoperatively, diuresis can be induced by an intravenous diuretic.

Absorption↗
PubMed1992

Isolation, characterization, and nucleotide sequence of the Streptococcus mutans mannitol-phosphate dehydrogenase gene and the mannitol-specific factor III gene of the phosphoenolpyruvate phosphotransferase system.

Streptococcus mutans, the causative agent of dental caries, utilizes carbohydrates by means of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTS facilitates vectorial translocation of metabolizable carbohydrates to form the corresponding sugar-phosphates, which are subsequently converted to glycolytic intermediates. The PTS consists of both sugar-specific and sugar-independent components. Complementation of an Escherichia coli mtlD mutation with a streptococcal recombinant DNA library allowed isolation of the mannitol-1-phosphate dehydrogenase gene (mtlD) and the adjacent sugar-specific mannitol factor III gene (mtlF) from S. mutans. Subsequent transposon mutagenesis of the complementing DNA fragment with Tn5seq1 defined the region that encodes the mtlD-complementing activity, the streptococcal mtlD gene. Nucleotide sequence analysis of this region revealed two complete open reading frames (ORFs) from within the streptococcal mannitol PTS operon. One ORF encodes the mtlD gene product, a 43.0-kDa protein which exhibits similarity to the E. coli and Enterococcus faecalis mannitol-1-phosphate dehydrogenases. The second ORF encodes a 15.8-kDa protein which exhibits similarity to mannitol factor III proteins from several bacterial species. In vitro transcription-translation assays were used to produce proteins of the sizes predicted by the streptococcal ORFs. These data indicate that the S. mutans mannitol PTS utilizes an enzyme II-factor III complex similar to the mannitol system found in other gram-positive organisms, as opposed to that of E. coli, which utilizes an independent enzyme II system.

Amino Acid Sequence↗
PubMed1984

In vitro basophil histamine release induced by mannitol in a patient with a mannitol-induced anaphylactoid reaction.

Anaphylactoid reactions in man are associated with the infusion of diagnostic and therapeutic agents. The etiology of such reactions remains unknown, but clinically they have the signs and symptoms that mimic those associated with in vivo histamine release. We report that the leukocytes of a patient who suffered an anaphylactoid reaction associated with the infusion of mannitol repeatedly released histamine in vitro when the leukocytes were challenged with low concentrations of mannitol (less than 0.1 M). In 44% deuterium oxide buffer, the patient's cells were tenfold more reactive to mannitol as compared to normal leukocytes and were pharmacologically modulated in a fashion similar to IgE-dependent release. The temperature optimum for this nonhyperosmolar (less than 0.1M) mannitol-induced, deuterium oxide-dependent release was the same as that for IgE-dependent release. Desensitization of the patient's cells with anti-IgE completely suppressed release induced by low concentrations of mannitol but did not affect the hyperosmolar (greater than 0.1M) mannitol-induced release of basophil histamine. These studies suggest that patients experiencing anaphylactoid reactions may be identified by use of in vitro basophil histamine release.

Anaphylaxis↗
PubMed1991

Cytoplasmic phosphorylating domain of the mannitol-specific transport protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli: overexpression, purification, and functional complementation with the mannitol binding domain.

The cytoplasmic C-terminal domain, residues 348-637, and the membrane-bound N-terminal domain, residues 1-347, of EIImtl have been subcloned and expressed in Escherichia coli. The N-terminal domain, IICmtl, contains the mannitol binding site, and the C-terminal domain, IIBAmtl, contains the activity-linked phosphorylation sites, His-554 and Cys-384. Overexpression of the BA domain was achieved by a translational in-frame fusion of the gene with the cro ATG start codon, downstream of the strong PR promoter of phage lambda. The domain has been purified and characterized in in vitro complementation assays. It possessed no mannitol phosphorylation activity itself but was able to restore the phosphoenolpyruvate-dependent phosphorylation activity of two EIImtl phosphorylation site mutants, lacking His-554 or Cys-384. The complementary N-terminal domain was also expressed. Membranes possessing IICmtl were unable to phosphorylate mannitol at the expense of phosphoenolpyruvate. However, when the membranes were combined with the purified C-terminal domain, mannitol phosphorylation activity was restored. Mannitol transport and phosphorylation were also restored in vivo when the two plasmids encoding the N- and C-terminal domains were expressed in the same cell. These data demonstrate the existence of structurally and functionally distinct domains in EIImtl: a cytoplasmic domain with phosphorylating activity and a membrane-bound N-terminal domain which, in the presence of the cytoplasmic domain, is able to actively transport and phosphorylate mannitol. The ability to separate, overproduce, and purify structurally stable, enzymatically active domains opens the way for 3D structural studies as well as complete kinetic analysis of the activities of the individual domains and their interactions.

Base Sequence↗
PubMed1991

Details of mannitol transport in Escherichia coli elucidated by site-specific mutagenesis and complementation of phosphorylation site mutants of the phosphoenolpyruvate-dependent mannitol-specific phosphotransferase system.

The mannitol transport protein (EIImtl) carries out translocation with concomitant phosphorylation of mannitol from the periplasm to the cytoplasm, at the expense of phosphoenolpyruvate (PEP). The phosphoryl group which is needed for this group translocation is sequentially transferred from PEP via two phosphorylation sites, located exclusively on the C-terminal cytoplasmic domain, to mannitol. Oligonucleotide-directed mutagenesis was used to investigate the precise role of these sites in phosphoryl group transfer, by producing specific amino acid substitutions. The first phosphorylation site, His-554 (P1), was replaced by Ala, which renders the EII-H554A completely inactive in PEP-dependent mannitol phosphorylation, but not in mannitol/mannitol 1-phosphate exchange. The P2 site mutant, EII-C384S, was inactive both in the mannitol phosphorylation reaction and in the exchange reaction, due to replacement of the essential Cys-384 by Ser. Although EII-H554A and EII-C384S were both catalytically inactive in the PEP-dependent phosphorylation, EII-C384S was able to restore up to 55% of the wild-type mannitol phosphorylation activity with the EII-H554A mutant, indicating a direct phosphotransfer between two subunits. These phosphorylation data together with the data obtained from mannitol/mannitol phosphate exchange kinetics, after mixing EII-H554A and EII-C384S, indicated the formation of functionally stable heterodimers, which consist of an EII-H554A and an EII-C384S monomer.

Base Sequence↗
PubMed1977

D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate.

Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.

Fungi↗
PubMed2004

Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

Genetic Engineering↗
PubMed1998

Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense.

Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungus Alternaria alternata in the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.

Journal Article↗
PubMed2003

Isovolume hypertonic solutes (sodium chloride or mannitol) in the treatment of refractory posttraumatic intracranial hypertension: 2 mL/kg 7.5% saline is more effective than 2 mL/kg 20% mannitol.

OBJECTIVE: To evaluate the clinical benefit of increasing the osmotic load of the hypertonic solution administered for the treatment of refractory intracranial hypertension episodes in patients with severe head injury. DESIGN: Prospective, randomized study. SETTINGS: A trauma center in a university hospital. PATIENTS: Twenty consecutive patients with head trauma and persistent coma who required infusions of an osmotic agent to treat episodes of intracranial hypertension resistant to well-conducted standard modes of therapy were studied. Intracranial hypertension was considered refractory when it persisted despite deep sedation, optimal hemodynamic status, and, in some patients, drainage of cerebral spinal fluid. INTERVENTIONS: Patients were randomly assigned to receive isovolume infusions of either 7.5% hypertonic saline solution (2400 mOsm/kg/H(2)O) or 20% mannitol (1160 mOsm/kg/H(2)O). The patients were given 2 mL/kg (body weight) of either solution, i.e., 361 +/- 13 mOsm of saline or 175 +/- 12 mOsm of mannitol per injection. MEASUREMENTS AND MAIN RESULTS: The main variables studied were the number and the duration of episodes of intracranial hypertension per day during the study period, which was stopped after the last episode of intracranial hypertension was recorded from intracranial pressure monitoring or after the allocated treatment failure. Patients in the HHS group were monitored for 7 +/- 5 days and those in the mannitol group for 7 +/- 6 days (not significant). The rate of failure for each treatment was also evaluated. Failure was defined as the persistence of intracranial hypertension despite two successive infusions of the same osmotic agent. The mean number of osmotic solute infusions was 3.7 +/- 5.3 in the mannitol group and 3.3 +/- 4.1 in the hypertonic saline solution group (not significant). The mean number (6.9 +/- 5.6 vs. 13.3 +/- 14.6 episodes) of intracranial hypertension episodes per day and the daily duration (67 +/- 85 vs. 131 +/- 123 min) of intracranial hypertension episodes were significantly lower in the hypertonic saline solution group (p <.01). The rate of clinical failure was also significantly lower in the hypertonic saline solution group: 1 of 10 patients vs. 7 of 10 patients (p <.01). CONCLUSION: In this study, when a hypertonic solute was required for the treatment of refractory intracranial hypertension episodes in patients with severe head trauma, increasing the osmotic load by giving 2 mL/kg (body weight) of 7.5% saline (361 +/- 13 mOsm) was more effective than giving 2 mL/kg (body weight) of 20% mannitol (175 +/- 12 mOsm). Within the limitations of the present study, these data suggest that giving 2 mL/kg hypertonic saline solution (approximately 480 mOsm/70 kg body weight) is an effective and safe initial treatment for intracranial hypertension episodes in head-trauma patients when osmotherapy is indicated.

Adult↗
PubMed1993

Expression and characterization of a structural and functional domain of the mannitol-specific transport protein involved in the coupling of mannitol transport and phosphorylation in the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli.

The mannitol-specific transport protein in Escherichia coli, EIImtl, consists of three structural and functional domains: a hydrophilic EIII-like domain (the A domain); a hydrophobic transmembrane domain (the C domain); and a second hydrophilic domain (the B domain) which connects the A and C domains together. The A domain contains the first phosphorylation site, His554, while the B domain contains the second phosphorylation site, Cys384. The phosphoryl group which is needed for the active transport of mannitol is sequentially transferred from P-enolpyruvate via the two phosphorylation sites to mannitol bound to the substrate binding site. In this paper, the expression, purification, and initial characterization of the B domain, IIBmtl, are described. Oligonucleotide-directed mutagenesis was used to produce an amber stop codon (TAG) and HindIII restriction site in a flexible loop between the B and A domains in the subcloned gene fragment coding for IIBAmtl (van Weeghel et al., 1991c). The gene fragment coding for IIBmtl was then subcloned behind strong promoters, located in two different expression/mutagenesis vectors, which directed the expression of the 15.3-kDa polypeptide in Escherichia coli. The domain was purified from E. coli crude cell extracts by using Q-Sepharose Fast Flow, S-Sepharose Fast Flow, and hydroxylapatite column steps. This purification procedure resulted in 1 mg of pure IIBmtl/g of cell, wet weight. The purified B domain was analyzed in vitro for its catalytic activity with membranes containing the phosphorylation site mutant form of EIImtl, C384S, and with the transmembrane domain, IICmtl. The B domain, together with purified IIA, was able to restore the P-enolpyruvate-dependent phosphorylation activity of the membrane-bound C domain. Steady-state mannitol phosphorylation kinetics at saturating EI, HPr, and IIAmtl yielded an apparent Km of P-IIBmtl for IICmtl of 200 microM and an apparent Vmax of 71 nmol of mtl-P min-1 mg of membrane protein)-1. This Vmax value is comparable to that of wild-type EIImtl measured under the same experimental conditions.

Base Sequence↗
PubMed2000

Multiple phosphorylation events regulate the activity of the mannitol transcriptional regulator MtlR of the Bacillus stearothermophilus phosphoenolpyruvate-dependent mannitol phosphotransferase system.

D-mannitol is taken up by Bacillus stearothermophilus and phosphorylated via a phosphoenolpyruvate-dependent phosphotransferase system (PTS). Transcription of the genes involved in mannitol uptake in this bacterium is regulated by the transcriptional regulator MtlR, a DNA-binding protein whose affinity for DNA is controlled by phosphorylation by the PTS proteins HPr and IICB(mtl). The mutational and biochemical studies presented in this report reveal that two domains of MtlR, PTS regulation domain (PRD)-I and PRD-II, are phosphorylated by HPr, whereas a third IIA-like domain is phosphorylated by IICB(mtl). An involvement of PRD-I and the IIA-like domain in a decrease in affinity of MtlR for DNA and of PRD-II in an increase in affinity is demonstrated by DNA footprint experiments using MtlR mutants. Since both PRD-I and PRD-II are phosphorylated by HPr, PRD-I needs to be dephosphorylated by IICB(mtl) and mannitol to obtain maximal affinity for DNA. This implies that a phosphoryl group can be transferred from HPr to IICB(mtl) via MtlR. Indeed, this transfer could be demonstrated by the phosphoenolpyruvate-dependent formation of [(3)H]mannitol phosphate in the absence of IIA(mtl). Phosphoryl transfer experiments using MtlR mutants revealed that PRD-I and PRD-II are dephosphorylated via the IIA-like domain. Complementation experiments using two mutants with no or low phosphoryl transfer activity showed that phosphoryl transfer between MtlR molecules is possible, indicating that MtlR-MtlR interactions take place. Phosphorylation of the same site by HPr and dephosphorylation by IICB(mtl) have not been described before; they could also play a role in other PRD-containing proteins.

Amino Acid Sequence↗
PubMed1988

Bacterial phosphoenolpyruvate-dependent phosphotransferase system: mannitol-specific EII contains two phosphoryl binding sites per monomer and one high-affinity mannitol binding site per dimer.

The amino acid composition and sequence of EIIMtl is known [Lee, C. A., & Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 10761-10767]. This information was combined, in the present study, with quantitative amino acid analysis to determine the molar concentration of the enzyme. The stoichiometry of phosphoryl group incorporation was then determined by phosphorylation of enzyme II from [14C]-phosphoenolpyruvate (pyruvate burst procedure). The native, reduced enzyme incorporated two phosphoryl groups per monomer. Both phosphoryl groups were shown to be transferred to mannitol. Oxidation or N-ethylmaleimide (NEM) labeling of Cys-384 resulted in incorporation of only one phosphoryl group per monomer, which was unable to be transferred to mannitol. The number of mannitol binding sites on enzyme II was determined by centrifugation using Amicon Centricon microconcentrators. The reduced unphosphorylated enzyme contained one high-affinity binding site (KD = 0.1 microM) per dimer and a second site with a KD in the micromolar range. Oxidation or NEM labeling did not change the number of binding sites.

Binding Sites↗
PubMed1992

[Mannitol solution (M-400) vs. mannitol solution combined with allopurinol in the prevention of post-transfusion acute renal failure].

Evaluation on the incidence of Acute Renal Failure (ARF) from a total of 41 corpse renal graft receptors, 20 of which received perfusion with mannitol solution at 400 mOsm/kg (Group 1) and 21 the same mannitol solution plus allopurinol (Group 2). No significant differences were present between both groups when they were analyzed for ARF incidence (15% vs 23.8%); overall incidence was 19.5%. 24-hour initial diuresis was shown to be an excellent predictor for long-term graft functionality. We can conclude that the addition of mannitol to the perfusion solution contributes decisively to reduce post-transplantation ARF, while addition of allopurinol does not have further beneficial effects.

Acute Kidney Injury↗
PubMed2005

Visualization of the phosphorylated active site loop of the cytoplasmic B domain of the mannitol transporter II(Mannitol) of the Escherichia coli phosphotransferase system by NMR spectroscopy and residual dipolar couplings.

The solution structure of a stably phosphorylated form of the cytoplasmic B domain of the mannitol-specific transporter (IIB(Mtl)) of the Escherichia coli phosphotransferase system, containing a mutation of the active site Cys384 to Ser, has been solved by NMR. The strategy employed relies principally on backbone residual dipolar couplings recorded in three different alignment media, supplemented by nuclear Overhauser enhancement data and torsion angle restraints related specifically to the active site loop (residues 383-393). As judged from the dipolar coupling data, the remainder of the structure is unchanged upon phosphorylation within the errors of the coordinates of the previously determined solution structure of unphosphorylated wild-type IIB(Mtl). Thus, only the active site loop was refined. Phosphorylation results in a backbone atomic rms shift of approximately 0.7 angstroms in the active site loop. The resulting conformation is less than 0.5 angstroms away from the equivalent P-loop in both the low and high molecular mass eukaryotic tyrosine phosphatases. 3J(NP) coupling constant measurements using quantitative J-correlation spectroscopy provide a direct demonstration of a hydrogen bond between the phosphoryl group and the backbone amide of Ser391 at position i + 7 from phospho-Ser384, with an approximately linear P-O-H(N) bond angle. The structure also reveals additional hydrogen bonding interactions involving the backbone amides of residues at positions i + 4 and i + 5, and the hydroxyl groups of two serine residues at positions i + 6 and i + 7 that stabilize the phosphoryl group.

Binding Sites↗