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PubMed2004

Thrombin stimulation of Cl-/HCO3- exchange in human platelets.

The presence of one acidifying Cl-/HCO3- exchange mechanism in human platelets has not been previously reported. This paper demonstrates that this mechanism does function and that it increases its activity after stimulation with thrombin. On resuspension of BCECF-loaded platelets in a chloride-free medium (gluconate replaced) that contains bicarbonate, cytosolic pH (pHi) increased and stabilized after 10 min at an alkaline value. After addition of 50 mM NaCl, pHi fell rapidly reaching steady state in the succeeding 5 min. The stilbene derivative 4-acetamido-4'-isothiocyanato stilbene-2,2' disulfonic acid (SITS) inhibited both, the alkalization in chloride-poor solution and the recovery from the alkaline load after chloride enrichment. The decline in pHi was observed whether chloride was delivered to the solution in the form of LiCl or NaCl, or when the later was applied after blockage of the Na+/H+ exchanger. The recovery in chloride-containing solution was in contrast to the effect of a similar change in osmolarity by addition of 50 mM sodium gluconate that did not produced a significant variation of pHi. Posterior addition of NaCl after 5 min in high gluconate reproduced the pHi fall of the control experiment. Alkali loads produced by 25 mM trimethylamine hydrochloride (TMA) were also counteracted by HCO(3-)-equivalent efflux via Cl-/HCO3- exchange. One of the major observations of the present study is that HCO3- equivalent efflux was twice as high when the platelets were previously stimulated with 0.1 IU of thrombin, but thrombin did not produce significant changes of the pHi recovery rate in a bicarbonate-free solution. The increase of the decline in pHi elicited by preexposure to thrombin was still observed in the presence of an inhibitor of the Na+/H+ exchange or in sodium-free solutions. It is concluded that a Na-independent Cl-/HCO3- exchange mechanism mediates the recovery of pHi from alkalosis in platelets and that thrombin activates this exchanger by a direct regulatory pathway.

Alkalosis↗
PubMed2003

Stimulation of Na+/Mg2+ antiport in rat erythrocytes by intracellular Cl-.

Mg(2+) efflux from rat erythrocytes was measured in NaCl, NaNO(3), NaSCN and Na gluconate medium. Substitution of extracellular and intracellular Cl(-) with the permeant anions NO(3)(-) and SCN(-) reduced Mg(2+) efflux via Na(+)/Mg(2+) antiport. After substitution of extracellular Cl(-) with the non-permeant anion gluconate, Mg(2+) efflux was not significantly reduced. In Na gluconate medium, an influence of the changed membrane potential and intracellular pH on Mg(2+) efflux could be excluded. The results indicate the existence of Cl(-)-independent Na(+)/Mg(2+) antiport and of Na(+)/Mg(2+) antiport stimulated by intracellular Cl(-). Intracellular Cl(-), as determined by means of (36)Cl(-), was found to stimulate Na(+)/Mg(2+) antiport through a cooperative effect according to a sigmoidal kinetics. The Hill coefficient for intracellular Cl(-) amounted to 1.4-1.8, indicating that two intracellular Cl(-) may be simultaneously active. With respect to specificity, Cl(-) was most effective, followed by Br(-), J(-), and F(-). Stimulation of Na(+)/Mg(2+) antiport by intracellular Cl(-) together with intracellular Mg(2+) may play a role during deoxygenation of erythrocytes and in essential hypertension.

Animals↗
PubMed2003

Safety in iron management.

Intravenous (IV) iron therapy has become an integral part of hemodialysis management during the past several decades, and the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative guidelines recognize that most patients undergoing hemodialysis will require IV iron therapy on a regular basis to reach target hemoglobin (Hgb) levels. There now are three IV iron compounds available in the United States: iron dextran, sodium ferric gluconate, and iron sucrose. Although all have been proven effective for increasing Hgb/hematocrit levels, recent data show differences in their relative safety profiles. During the past two decades, more than 30 deaths have been attributed to the use of IV iron dextran. The two newer compounds available in the United States, sodium ferric gluconate and iron sucrose, have more favorable safety profiles, with the largest prospective safety comparison to date showing sodium ferric gluconate to be similar to placebo in the incidence of serious anaphylactoid-type reactions. This article reviews safety data surrounding the IV iron therapies.

Anaphylaxis↗
PubMed2003

The glyoxylate bypass of Ralstonia eutropha.

The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli. Interposon-mutants of all three genes (DeltaaceA1, DeltaaceA2 and DeltaaceB1) were constructed, and the phenotypes of the respective mutants were investigated. Whereas R. eutropha HF39DeltaaceA1 retained only 19% of ICL activity and failed to grow on acetate, R. eutropha HF39DeltaaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded. These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle. R. eutropha HF39DeltaaceB1 retained on acetate as well as on gluconate about 41-42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R. eutropha HF39. Whereas in R. eutropha HF39DeltaaceA1 and R. eutropha HF39DeltaaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R. eutropha HF39DeltaaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R. eutropha HF39.

Acetates↗
PubMed1998

Systemic and topical effects of intradermal hydrofluoric acid.

A 35-year-old man attempted suicide by subcutaneous self-administration of hydrofluoric acid (5 cc of a domestic rust removal solution containing 7% hydrofluoric acid). A burn 9 x 7 cm in diameter immediately appeared at the injection site (left forearm and fold of the elbow). In the emergency department, the burn was copiously washed with isotonic solution and treated with cutaneous and subcutaneous injections of magnesium chloride, 10% solution of calcium gluconate, and 2% xylocaine, then continuously maintained under topical treatment with calcium gluconate. Seven hours after injection, the patient was severely hypocalcemic (Ca+2 0.64 mmol/L). Ten hours after injection, in addition to the persistent hypocalcemia (Ca+2 0.81 mmol/L), hyponatremia (123 mmol/d), hypokalemia (3.4 mmol/L), and hypochloremia (95.6 mmol/L) had developed. The hypocalcemia was corrected with infusion of calcium gluconate (8.92 mEq of Ca+2 as total amount). The patient underwent surgical intervention 7 days after admission, followed by several interventions of plastic surgery.

Adult↗
PubMedDate not supplied

Contaminated anterior cruciate ligament grafts: the efficacy of 3 sterilization agents.

PURPOSE: A study was undertaken to determine the incidence of positive cultures resulting from an anterior cruciate ligament (ACL) specimen dropped on the operating room floor and the efficacy of sterilizing the specimen by soaking in 1 of 3 antimicrobial solutions: an antibiotic solution of neomycin and polymyxin B, 10% providone-iodine solution, and standard chlorhexidine gluconate solution. TYPE OF STUDY: Randomized trial. MATERIALS AND METHODS: Fifty ACL specimens removed from patients undergoing total knee arthroplasty were used as the test group. The specimens were longitudinally sectioned into 4 equal pieces. The 4 pieces were dropped on the floor and left for a period of 15 seconds. Cultures were taken from each specimen after immersion in 1 of the 3 sterilization solutions for a period of 90 seconds. One of the 4 specimens was cultured without being exposed to any solution, thereby establishing these specimens as the control group. Cultures of a floor swab were taken at the same time and place that the ACL was dropped. RESULTS: The floor swab cultures were positive in 48 of the 50 specimens (96%). The ACL control group (untreated dropped grafts) had 29 of 50 specimens positive (58%). The grafts soaked in antibiotic solution had 3 of 50 specimens positive (6%). The grafts soaked in providone-iodine solution had 12 of 50 specimens positive (24%). The grafts soaked in chlorhexidine gluconate solution had 1 of 50 specimens positive (in broth only) (2%). CONCLUSION: This study shows that significant contamination occurs when dropping specimens on the floor, as 58% of the dropped grafts had positive cultures. Of the 3 sterilization techniques used, chlorhexidine gluconate seems to be the most efficient with only a single broth culture (2%) found to be positive. The antibiotic solution was second best (6%), although there is no statistically significant difference between these 2 groups. The 10% providone-iodine solution under these test conditions was the least effective of all the 3 sterilization agents with 24% cultures positive after immersion.

Anterior Cruciate Ligament↗
PubMed1985

Efficacy of low-concentration iodophors for germicidal hand washing.

The efficacy of iodophor germicides containing different concentrations of available iodine against transient (inoculated) bacteria and the natural hand microflora was compared with chlorhexidine gluconate (2 and 4%) liquid detergent (Hibitane), non-germicidal soap and a tap water rinse. The tap water rinse was ineffective compared with all other treatments. Only 4% chlorhexidine gluconate liquid detergent and iodophor containing 0.75% available iodine were significantly better than the non-germicidal soap for reduction of transient bacteria, Escherichia coli and Pseudomonas fluorescens, that had been inoculated onto hands. These agents also caused a significant reduction in the number of 'natural' microorganisms released from hands after a standard 15 s hand wash. The low-concentration iodophor products and the product containing 2% chlorhexidine gluconate failed to give results significantly better than the non-germicidal control soap. Baird-Parker medium and standard aerobic plate counts were highly correlated (r = 0.82), so that for studies of Gram-negative bacteria inoculated onto hands as a transient microflora, counts on Baird-Parker medium give a reasonable indication of the natural (residual) hand microflora.

Chlorhexidine↗
PubMedDate not supplied

Continuous production of high-content fructooligosaccharides by a complex cell system.

A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module was employed to produce high-content fructooligosaccharides (FOS) in a continuous process initiated by a batch process. The system used mycelia of Aspergillus japonicus CCRC 93007 or Aureobasidium pullulans ATCC 9348 with beta-fructofuranosidase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity. Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate. Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high-content FOS was discharged continuously from a MF module. The complex cell system was run at 30 degrees C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5-7% glucose and 8-10% sucrose on a dry weight basis, plus a small amount of calcium gluconate. The system worked for a 7-day continuous production process with a dilution rate of 0.04 h(-1), and the volumetric productivity for total FOS was more than 160 g L(-1) h(-1).

Ascomycota↗
PubMed2006

Tetranuclear copper(II) complexes bridged by alpha-D-glucose-1-phosphate and incorporation of sugar acids through the Cu4 core structural changes.

Tetranuclear copper(II) complexes containing alpha-D-glucose-1-phosphate (alpha-D-Glc-1P), [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(bpy)4(H2O)2]X3 [X = NO3 (1a), Cl (1b), Br (1c)], and [Cu4(mu-OH){mu-(alpha-D-Glc-1P)}2(phen)4(H2O)2](NO3)3 (2) were prepared by reacting the copper(II) salt with Na2[alpha-D-Glc-1P] in the presence of diimine ancillary ligands, and the structure of 2 was characterized by X-ray crystallography to comprise four {Cu(phen)}2+ fragments connected by the two sugar phosphate dianions in 1,3-O,O' and 1,1-O mu4-bridging fashion as well as a mu-hydroxo anion. The crystal structure of 2 involves two chemically independent complex cations in which the C2 enantiomeric structure for the trapezoidal tetracopper(II) framework is switched according to the orientation of the alpha-D-glucopyranosyl moieties. Temperature-dependent magnetic susceptibility data of 1a indicated that antiferromagnetic spin coupling is operative between the two metal ions joined by the hydroxo bridge (J = -52 cm(-1)) while antiferromagnetic interaction through the Cu-O-Cu sugar phosphate bridges is weak (J = -13 cm(-1)). Complex 1a readily reacted with carboxylic acids to afford the tetranuclear copper(II) complexes, [Cu4{mu-(alpha-D-Glc-1P)}2(mu-CA)2(bpy)4](NO3)2 [CA = CH3COO (3), o-C6H4(COO)(COOH) (4)]. Reactions with m-phenylenediacetic acid [m-C6H4(CH2COOH)2] also gave the discrete tetracopper(II) cationic complex [Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)(CH2COOH))2(bpy)4](NO3)2 (5a) as well as the cluster polymer formulated as {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-m-C6H4(CH2COO)2)(bpy)4](NO3)2}n (5b). The tetracopper structure of 1a is converted into a symmetrical rectangular core in complexes 3, 4, and 5b, where the hydroxo bridge is dissociated and, instead, two carboxylate anions bridge another pair of Cu(II) ions in a 1,1-O monodentate fashion. The similar reactions were applied to incorporate sugar acids onto the tetranuclear copper(II) centers. Reactions of 1a with delta-D-gluconolactone, D-glucuronic acid, or D-glucaric acid in dimethylformamide resulted in the formation of discrete tetracopper complexes with sugar acids, [Cu4{mu-(alpha-D-Glc-1P)}2(mu-SA)2(bpy)4](NO3)2 [SA = D-gluconate (6), D-glucuronate (7), D-glucarateH (8a)]. The structures of 6 and 7 were determined by X-ray crystallography to be almost identical with that of 3 with additional chelating coordination of the C-2 hydroxyl group of D-gluconate moieties (6) or the C-5 cyclic O atom of D-glucuronate units (7). Those with D-glucaric acid and D-lactobionic acid afforded chiral one-dimensional polymers, {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-D-glucarate)(bpy)4](NO3)2}n (8b) and {[Cu4{mu-(alpha-D-Glc-1P)}2(mu-D-lactobionate)(bpy)4(H2O)2](NO3)3}n (9), respectively, in which the D-Glc-1P-bridged tetracopper(II) units are connected by sugar acid moieties through the C-1 and C-6 carboxylate O atoms in 8b and the C-1 carboxylate and C-6 alkoxy O atoms of the gluconate chain in 9. When complex 7 containing d-glucuronate moieties was heated in water, the mononuclear copper(II) complex with 2-dihydroxy malonate, [Cu(mu-O2CC(OH)2CO2)(bpy)] (10), and the dicopper(II) complex with oxalate, [Cu2(mu-C2O4)(bpy)2(H2O)2](NO3)2 (11), were obtained as a result of oxidative degradation of the carbohydrates through C-C bond cleavage reactions.

Carboxylic Acids↗
PubMed1999

Screening for L-sorbose and L-sorbosone dehydrogenase producing microbes for 2-keto-L-gulonic acid production.

Acetic acid bacteria incompletely oxidize L-sorbose to 2-keto-L-gulonic acid (2KLG) by L-sorbose- and L-sorbosone dehydrogenases. In order to isolate novel microorganisms with these enzyme activities, a new screening method has been studied with a presumption that microorganisms reuse their metabolic products when principal carbon sources are exhausted. When various keto-aldonic acid-producing microorganisms were tested for the ability to grow in minimal media containing such products as 2,5-diketo-gluconic acid, 2-keto-D-gluconic acid, 5-keto-D-gluconic acid or 2-keto-L-gulonic acid, they grew with these keto-aldonic acids as the sole carbon source. By enriching the isolates collected from screening samples for their growth in minimal medium containing 2KLG as the sole carbon source, as much as 50% of selected strains showed L-sorbose- and L-sorbosone dehydrogenase activities. In spite of the presence of these enzymes, no significant amount of 2KLG was detected in the culture broth, possibly due to 2KLG reductase activity, indicating that the direct screening for 2KLG producer microorganisms would be less successful. These results suggest that the screening strategy using 2KLG as a carbon source is a useful method for the selective screening of microorganisms with L-sorbose- and L-sorbosone dehydrogenases, and that a similar strategy may be applied to other cases.

Journal Article↗
PubMed2002

Corrosion rates of stainless-steel files in different irrigating solutions.

AIM: The present study was designed to evaluate and compare the corrosion rates of the stainless-steel endodontic files when immersed in 0.2% chlorhexidine gluconate, 5.25% NaOCl, chlorinated soda with KOH, and 17% EDTA irrigating solutions. METHODOLOGY: Corrosion rates of stainless-steel K-files in irrigating solutions were determined electrochemically by the Tafel extrapolation method. The cutting flutes of files were immersed in solutions and used as an electrode. A saturated calomel electrode (SCE) was used as a reference, and a platinum plate was used as a counter electrode. In order to determine corrosion rates, the linear part of anodic currents obtained from electrochemical current-potential curves, was extrapolated to corrosion potentials. RESULTS: The corrosion rates of stainless-steel files in the tested solutions from the highest to the lowest were: 0.2% chlorhexidine gluconate>5.25% NaOCl>chlorinated soda with KOH>17% EDTA. The differences between the corrosion rates of files were not found to be statistically different for chlorinated soda with KOH and EDTA (P >0.05), but statistically significant differences were found amongst other groups. CONCLUSIONS: The results of the present study indicated that 0.2% chlorhexidine gluconate, 5.25% NaOCl and chlorinated soda with KOH cause severe corrosion on the surface of selected stainless-steel files.

Analysis of Variance↗
PubMed2003

Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro.

AIM: To evaluate the effectiveness of 2% chlorhexidine gluconate gel and calcium hydroxide (Ca(OH)2) as intracanal medicaments against Enterococcus faecalis. METHODOLOGY: One hundred and eighty dentine tubes prepared from intact freshly extracted bovine maxillary central incisors were infected in vitro for 7 days with E. faecalis. The specimens were divided into four groups, according to the intracanal medicament used, as follows: Group 1: 2% chlorhexidine gluconate gel; Group 2: calcium hydroxide in a viscous vehicle (polyethyleneglycol 400); Group 3: 2% chlorhexidine gluconate gel + calcium hydroxide and Group 4: Brain Heart Infusion (BHI) broth (control group). The medicaments were placed into the canal lumen and left there for experimental times of 1, 2, 7, 15 and 30 days. After each period, irrigation with sterile saline to remove the medicament was performed and the canals were dried with sterile paper points. Dentine chips were removed from the canals with sequential sterile round burs at low speed. The samples obtained with each bur were immediately collected in separate test tubes containing BHI broth. The tubes were incubated at 37 degrees C and daily observed for microbial growth, visualized by the medium turbidity. RESULTS: Chlorhexidine gel alone completely inhibited the growth of E. faecalis after 1, 2, 7 and 15 days. Calcium hydroxide allowed microbial growth at all experimental times. The combination of chlorhexidine and Ca(OH)2 was effective after 1 and 2 days demonstrating 100% antibacterial action; however, its antibacterial activity reduced between 7 and 15 days. CONCLUSION: Under the conditions of this study, it can be concluded that 2% chlorhexidine gel alone was more effective against E. faecalis than calcium hydroxide (P < 0.05). However, its antibacterial activity depended on how long it remained inside the root canal.

Animals↗
PubMed2003

Controversies in iron management.

BACKGROUND: Iron therapy is required in hemodialysis patients receiving erythropoietic stimulators in order to achieve the target hemoglobin in the most efficient way. While oral iron has been disappointing in this regard, parenteral iron has been widely used, despite a significant incidence of severe side effects when iron dextran is used. The recent availability of a more effective form of oral iron (heme-iron), and safer forms of parenteral iron (iron sucrose and iron gluconate) has made iron management in this population simpler. Many questions remain, however, about the use, efficacy, and safety of these compounds in hemodialysis patients. METHODS: Current literature was reviewed and combined with the authors' clinical experience to address a number of current questions regarding the use of iron in hemodialysis patients. RESULTS: Although oral non-heme iron is infrequently sufficient to maintain iron stores in hemodialysis patients, recent studies suggest that heme-iron may be more useful in this regard. Heme-iron is absorbed to a greater extent than non-heme iron, and is better tolerated. Small studies have shown that when heme-iron is administered, less parenteral iron and lower doses of erythropoietin (EPO) are needed to maintain target hemoglobin. Current evidence suggests that both iron sucrose and iron gluconate are safer than iron dextran, and the latter should only be used in extraordinary circumstances. While in vitro studies have demonstrated some differences in the effects of iron sucrose and iron gluconate on cellular toxicity, the clinical importance of these has not been determined. Both compounds can be used safely for repletion and maintenance therapy, and doses of up to 300 mg of either are generally well tolerated when such higher doses are needed, as in peritoneal dialysis (PD) patients or chronic kidney disease (CKD) patients not on dialysis. CONCLUSION: A number of questions remain regarding the appropriate use, efficacy, and potential toxicity of iron therapy in dialysis patients. Further prospective research should address the myriad questions raised in this review.

Anemia↗
PubMed2005

Trends in intravenous iron use among dialysis patients in the United States (1994-2002).

BACKGROUND: Two new intravenous (IV) iron products, ferric gluconate and iron sucrose, recently were approved for use in the United States. We report trends in IV iron use in both incident (1994 to 2001) and prevalent (1994 to 2002) Medicare US dialysis patients. METHODS: Included patients had Medicare as a primary payer. Recombinant human erythropoietin doses, IV iron use, and hemoglobin data were obtained from Medicare outpatient files. The most recent cohorts included 241,770 prevalent hemodialysis (HD) patients in 2002 and 11,744 incident HD patients in 2001. RESULTS: For incident HD patients in the first 9 months of dialysis therapy, the percentage of patients administered IV iron increased sharply between 1994 and 1997 and then increased gradually between 1997 and 2001. In 2002, a total of 84.4% of HD and 19.3% of peritoneal dialysis (PD) patients were administered IV iron. Ferric gluconate use increased slowly in 2000, increased from 5.7% to 18.6% from December 2000 to January 2001, increased to 29.8% in April 2002, and was 23.3% in December 2002. Iron sucrose use increased to 26% by December 2002. The absolute monthly percentage of HD patients administered IV iron dextran decreased from 49.6% in January 2000 to 3.6% in December 2002. CONCLUSION: In US patients with end-stage renal disease, IV iron use has increased, although slowly, from 1997 to 2002. Ferric gluconate and iron sucrose have become the predominant form of therapy. IV iron therapy was used in a much smaller percentage of PD compared with HD patients, and racial and geographic variability was observed.

Adolescent↗
PubMed1981

Potential for selection among nearly neutral allozymes of 6-phosphogluconate dehydrogenase in Escherichia coli.

Six gnd alleles coding for naturally occurring allozymes of 6-phosphogluconate dehydrogenase [6-phospho-D-gluconate:NAD(P)+ 2-oxidoreductase, EC 1.1.1.43] have been transferred by transduction into the genetic background of Escherichia coli K-12 and examined for their selective effects in chemostats in which gluconate was limiting. Four of the alleles are evidently neutral or nearly neutral, inasmuch as their selective effects, if any, fall below the limit of resolution of the procedure--0.5%/hr or about 1% per generation. One allele is detrimental in limiting gluconate but not in limiting glucose or fructose. Another allele has a detrimental, density-dependent, epistatic interaction with tonA. We suggest that all six alleles are neutral or nearly neutral in natural populations but that they are not functionally equivalent; their functional differences are potentially important because they can become expressed as differences in fitness under the appropriate conditions of environment or genetic background. Under these conditions, otherwise neutral alleles can become subject to selection.

Alleles↗
PubMed2003

Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase.

The hyperthermophilic Archaeon Sulfolobus solfataricus metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to 2-keto-3-deoxygluconate. 2-Keto-3-deoxygluconate (KDG) aldolase then catalyzes the cleavage of 2-keto-3-deoxygluconate to glyceraldehyde and pyruvate. The gene encoding glucose dehydrogenase has been cloned and expressed in Escherichia coli to give a fully active enzyme, with properties indistinguishable from the enzyme purified from S. solfataricus cells. Kinetic analysis revealed the enzyme to have a high catalytic efficiency for both glucose and galactose. KDG aldolase from S. solfataricus has previously been cloned and expressed in E. coli. In the current work its stereoselectivity was investigated by aldol condensation reactions between D-glyceraldehyde and pyruvate; this revealed the enzyme to have an unexpected lack of facial selectivity, yielding approximately equal quantities of 2-keto-3-deoxygluconate and 2-keto-3-deoxygalactonate. The KDG aldolase-catalyzed cleavage reaction was also investigated, and a comparable catalytic efficiency was observed with both compounds. Our evidence suggests that the same enzymes are responsible for the catabolism of both glucose and galactose in this Archaeon. The physiological and evolutionary implications of this observation are discussed in terms of catalytic and metabolic promiscuity.

Aldehyde-Lyases↗
PubMed2004

The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner-Doudoroff pathway in Sulfolobus solfataricus.

The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 degrees C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to D-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to D-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and D-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and D-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and D-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 degrees C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus D-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.

Aldehyde-Lyases↗
PubMed2000

Anion permeation in Ca(2+)-activated Cl(-) channels.

Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore.

Animals↗