Presence Of Gluconate Research Articles

Buffer-dependent pH sensitivity of the fluorescent chloride-indicator dye SPQ.

The fluorescence intensity of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) in an N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 2-(N-morpholino)ethanesulfonic acid (MES)-Tris(hydroxymethyl)aminomethane buffer, pH 7.0, decreased as a function of Cl- concentration and/or gluconate concentration, as expected. Contrary to expectation, however, the fluorescence intensity progressively increased as the pH decreased, independently of the presence of gluconate. Consequently, the modulation of SPQ fluorescence by commonly used buffers was investigated as a function of pH. Titration curves demonstrated SPQ quenching and yielded pK values characteristic of each buffer. from here, pH-independent Stern-Volmer constants, KQbase, were calculated for each of the morpholine derivatives, MES and 3-(N-morpholino)-2-hydroxypropanesulfonic acid. In contrast, HEPES and piperazine-N,N'-bis(2-ethanesulfonic acid), which are piperazine derivatives, exhibited an additional pH-independent "molecular" quenching constant KmQ throughout the pH range 3-10. To study chloride fluxes, therefore, what counts is the apparent Cl-Stern-Volmer constant KappCl, which is a function of both pH and buffer composition. Equations describing these relationships are presented. In conclusion, unless both pH and the buffer composition are taken into account, SPQ is unsuitable for studying the concomitant transmembrane fluxes of Cl- and H+.

Regulation of the renal Na-HCO3 cotransporter by cAMP and Ca-dependent protein kinases.

Changes in the activity of the brush-border Na-H antiporter are accompanied by parallel changes in the activity of the Na-HCO3 cotransporter. Adenosine 3',5'-cyclic monophosphate (cAMP) and calmodulin inhibit the Na-H antiporter, whereas protein kinase C (PKC) stimulates it. We hypothesized that cAMP, calmodulin, and PKC should have similar effects on the Na-HCO3 cotransporter activity. Phosphorylated renal basolateral membranes were treated with either cAMP, calmodulin, or phorbol ester. cAMP, 1 microM, inhibited HCO3-dependent 22Na uptake without affecting 22Na uptake in presence of gluconate, suggesting that cAMP inhibits Na-HCO3 cotransporter activity without altering diffusive 22Na uptake. The effect of cAMP to inhibit the Na-HCO3 cotransporter could also be elicited by the catalytic subunit of cAMP, and this inhibitory effect was prevented by the protein kinase A (PKA) inhibitor. Calmodulin (1 microM), in presence of Ca, also inhibited HCO3-dependent 22Na uptake in presence of HCO3, whereas 22Na uptake in the presence of gluconate was unchanged. The inhibitory effect of calmodulin on HCO3-dependent 22Na uptake was prevented by N-(4-aminobutyl)-5-chloro-2-naphthalene sulfonamide (W-13), an inhibitor of calmodulin. Phorbol 12-myristate 13-acetate and PKC stimulated Na-HCO3 cotransporter activity, whereas the inactive analogue, 4 alpha-phorbol, failed to elicit such a stimulation. Basolateral membranes displayed cAMP-dependent and Ca-dependent protein kinase activities. Thus PKA and Ca-dependent protein kinases regulate the activity of the Na-HCO3 cotransporter and suggest that hormones that act through these systems modulate the activity of the Na-HCO3 cotransporter.

Glycolysis and Entner-Doudoroff pathways in Halobacterium halobium: Some new observations based on 13C NMR spectroscopy

13C NMR was used to study glucose metabolism in intact cells of Halobacterium halobium. Spectra of glucose grown cells incubated with [1-13C] glucose indicate the presence of gluconate as the initial product. The existence of glycolytic pathway is also indicated. In the extracts of these cells an NADP dependent glucose dehydrogenase was detected. Galactose grown cells failed to metabolise glucose but exhibited glucose dehydrogenase activity although about 20-50% less than that for glucose grown cells. Possible explanations of these experiments are discussed.

Formation of polyesters consisting of medium-chain-length 3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent pseudomonads

Pseudomonas aeruginosa PAO and 15 other strains of this species synthesized a polyester with 3-hydroxydecanoate as the main constituent (55 to 76 mol%) if the cells were cultivated in the presence of gluconate and if the nitrogen source was exhausted; 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydodecanoate were minor constituents of the polymer. The polymer was deposited in granules within the cell and amounted to 70% of the cell dry matter in some strains. Among 55 different strains of 41 Pseudomonas species tested, P. aureofaciens (21.6% of cellular dry matter), P. citronellolis (78.0%), P. chlororaphis (8.5%), P. marginalis (11.4%), P. mendocina (50.7%), P. putida (33.5%), and Pseudomonas sp. strain DSM 1650 (54.6%) accumulated this type of polymer at significant levels (greater than 5%) during cultivation on gluconate. In two strains of P. facilis and P. fluorescens, as well as in one strain of P. syringae, this polymer was detected as a minor constituent (much less than 5%). All other strains accumulated either poly(3-hydroxybutyrate) or a polymer consisting mainly of 3-hydroxyoctanoate with octanoate but no polyester with gluconate as the carbon source. Only a few species (e.g., P. stutzeri) were unable to accumulate poly(hydroxyalkanoic acids) (PHA) at all. These results indicated that the formation of PHA depends on a pathway which is distinct from all other known PHA-biosynthetic pathways. The polyesters accumulated by gluconate- or octanoate-grown cells of recombinant strains of P. aeruginosa and P. putida, which harbored the Alcaligenes eutrophus poly(3-hydroxybutyrate)biosynthetic genes, contained 3-hydroxybutyrate as an additional constituent.

Effect of mutations causing gluconate kinase or gluconate permease deficiency on expression of the Bacillus subtilis gnt operon

The gluconate (gnt) operon contains genes for a repressor of the operon, gluconate kinase, and gluconate permease. A nonleaky kinase mutation (gntK4) induced the gnt operon constitutively through interaction of the repressor with an inducer of gluconate which had been endogenously formed and accumulated in the cell owing to the complete deficiency of the kinase even in the absence of gluconate in the medium. In contrast, a nonleaky permease mutation (gntP9) never induced the operon by gluconate likely because it cannot give rise to its inducing concentration in the cell even in the presence of gluconate in the medium.

Modulation of ATP-dependent Ca 2+ transport in rat parotid basolateral membrane vesicles by K + + Cl − flux

In basolateral membrane vesicles (BLMV) isolated from rat parotodi glands, the initial rate of ATP-dependent Ca + transport, in the presence of KCl, was approx. 2-fold higher than that obtained with mannitol, sucrose or N- methyl- d-glucamine (NMDG)-gluconate. Only NH f4 4, Rb +, or Br − could effectively substitute for K + or Cl −, respectively. This KCl activation was concentration dependent, with maximal response by 50 mM KCl. An inwardly directed KCl gradient up to 50 mM KCl had no effect on Ca + transport, while equilibration of the vesicles with KCl (> 100 mM) increased transport 15–20%. In presence of Cl −, 86Rb + uptake was 2.5-fold greater than in the presence of gluconate. 0.5 mM furosemide inhibited 86Rb + flux by approx. 60% in a Cl − medium and by approx. 20% in a gluconate medium. Furosemide also inhibited KCl activation of Ca + transport with half maximal inhibition either at 0.4 mM or 0.05 mM, depending on whether 454Ca + transport was measured with KCl (150 mM) equilibrium or KCl (150 mM) gradient. In a mannitol containing assay medium, potassium gluconate loaded vesicles had higher (approx. 25%) rate of Ca + transport than mannitol loaded vesicles. Addition of valinomycin (5 μM) to potassium gluconate loaded vesicles further stimulated (approx. 30%) the Ca + transport rate. These results suggest that during ATP dependent Ca + transport in parotid BLMV, K + can be recycled by the concerted activities of a K + and Cl − coupled flux and a K + conductance.

Sulfate transport in apical membrane vesicles isolated from tracheal epithelium.

Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated system. This conclusion is based on three lines of evidence: 1) saturation kinetics; 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media (apparent Km = 0.13 mM) and decreases in the presence of gluconate (apparent Km = 0.68 mM). Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO3-, S2O3(2-), SeO4(2-), and MoO4(2-) but not by H2PO4- or HAsO4(2-). Studies indicate that protons may play two distinct roles in sulfate transport in this system. 1) Their possible modifier role is suggested by the fact that protons affect SO2-4 transport in an uncompetitive manner. 2) The possibility that the proton gradient may act as an energy source for a secondary active transport is indicated by the fact that the imposition of a proton gradient stimulates a transient movement of sulfate in to the tracheal apical membrane vesicle, against its concentration gradient, causing an "overshoot" phenomenon. Our studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient (OH- gradient) indicates that under those conditions the mechanism of transport may be a SO4(2-)-OH- exchange. The fact that chloride cis-inhibits and trans-stimulates SO4(2-) transport indicates that SO2-4 uptake may also occur via a SO4(2-)-Cl- exchange. Studies carried out so far do not enable us to conclude unequivocally whether the tracheal apical membrane system displays two distinct carrier activities (SO4(2-)-Cl-; SO4(2-)-OH-) or one anion exchanger, which like the erythrocyte anion exchanger, may interact with SO4(2-), Cl-, and H+. The fact that the anion transport inhibitors DIDS and SITS inhibit SO4(2-) transport in the presence or absence of chloride suggests that the latter possibility may be the case.

Mechanism of coupling between Cl − and OH − transport in renal brush-border membranes

The coupling mechanism for Cl − and H +/OH − transport in renal brush-border vesicles was examined from intravesicular pH changes following imposed H + and Cl − gradients. Vesicles were loaded with 6-carboxyfluorescein and exposed to H + gradients and Cl −, gluconate, or sulfate gradients, each with and without a K +/valinomycin voltage clamp. Parallel experiments were performed with vesicles equilibrated with 10 mM HCO 3 − or 5 mM formate. Rate of H +/OH − transport was determined from the initial rate of change in 6-carboxyfluorescein fluorescence, vesicle buffer capacity and the relationship between fluorescence and vesicle pH. In contrast to gluconate or sulfate, Cl − caused enhanced H +/OH − transport under all conditions. This difference was eliminated with voltage clamping in the presence of gluconate, SO 4 2−, or HCO 3 −, but not in the presence of formate. These findings were not affected by the method of preparation of the vesicles. Electrically coupled Cl −/OH − transport was not inhibited by 100 μM DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonate) or 100 μM DBDS (4,4′-dibenzamidostilbene-2,2′-disulfonate). SITS (4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate) was found to be a protonphore at concentrations > 500 μM. As a control for the method, we demonstrated amiloride inhibitable, electroneutral Na +-H + exchange (H + flux = 107 ± 9 nmol/s per mg, 100 mM Na +) and electroneutral, DBDS inhibitable Cl −-HCO 3 − exchange in sealed human red blood cell ghosts. Therefore, electroneutral Cl −-OH − or HCO 3 − exchange does not measurably contribute to Cl − transport in the proximal tubule brush border. Cl −-formate exchange with formic acid recyling appears to be the only electroneutral coupling mechanism between Cl − and OH − transport demonstrable in renal brush-border membrane vesicles.

Beta-Amino acid transport across the renal brush-border membrane is coupled to both Na and Cl.

Sodium-dependent beta-alanine uptake into dog renal brush-border membrane vesicles was studied. Kinetic analysis indicated a single transport system, highly specific for beta-amino acids, with Km = 35 microM at 100 mM NaCl. Sodium-dependent beta-alanine transport was markedly anion-dependent, being highest in the presence of chloride (Cl greater than Br greater than SCN greater than NO3 approximately I greater than F) and virtually nonexistent in the presence of gluconate and other nonphysiological chloride substitutes. In addition, it was observed that beta-alanine uptake could be driven against a concentration gradient by a chloride gradient. Similar results were found for sodium. Taken together, these observations provide strong evidence that beta-alanine transport across the renal brush-border membrane is coupled to both sodium and chloride. Studies of the dependence of beta-alanine flux on chloride and sodium concentrations indicated that one chloride ion and multiple sodium ions were involved in the beta-alanine transport event. beta-Alanine flux on chloride found to involve the net transfer of positive charge, consistent with these stoichiometric assignments. The hallucinogen harmaline inhibited beta-alanine uptake in a 1:1 fashion, presumably by acting at a single site on the transport molecule. The ability of harmaline to inhibit beta-alanine uptake was decreased when the chloride concentration was lowered but was unchanged when the sodium concentration was decreased. These results indicate that harmaline does not compete with sodium for a binding site on the carrier as has been suggested for other sodium-coupled transport systems, and that instead, chloride may be required for harmaline binding to the beta-alanine transporter.

Plasma membrane proton-ATPase of a turtle bladder epithelial cell line.

Urinary acidification by the turtle bladder is mediated by a proton ATPase located in the apical membrane. The present study describes a proton ATPase in the plasma membrane of a cell line of turtle bladder epithelial cells. In the presence of ouabain to inhibit Na+,K+-ATPase and in the absence of Ca2+ to inhibit Ca2+-ATPase, we measured ATPase activity of the plasma membranes of the cultured cells. This ATPase was resistant to oligomycin but sensitive to dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate. In the presence of ATP, the ATPase was capable of acidification as assessed by quenching of acridine orange. Acidification could not be elicited by other nucleotides (GTP, UTP). Acidification was inhibited by dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate but was not affected by replacement of Na+ by K+. The acidification response was dependent on the presence of chloride, abolished in the presence of gluconate, and inhibited partially by nitrate. Experiments utilizing the voltage-sensitive dye 3,3'-dipropylthiodicarbocyanine iodide showed that the proton ATPase was electrogenic and capable of responding to a favorable electric gradient. In summary, the turtle bladder epithelial cell line has a plasma membrane proton ATPase which is similar to the proton ATPase of turtle bladder epithelium and thus should allow purification and characterization of this enzyme.

Saturation behavior of ascites tumor cell chloride exchange in the presence of gluconate

Steady state Cl − flux across the Ehrlich mouse ascites cell membrane was studied when gluconate replaced Cl − in the external medium. Saturation behavior was observed; K 1 2 was 23.9 mM Cl − and V was 758 μmol · g −1 dry weight · h −1. The cells lost K +, Cl − and H 2O, consistent with relative impermeability to gluconate, and the Cl − efflux rate coefficient was elevated. The results indicate that a major portion of Cl − exchange occurs as a membrane transport process and suggest that the process is sensitive to intracellular Cl − levels.

Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia, and Klebsiella species

Various enterobacteria exhibit several patterns of synthesis and excretion of polygalacturonic acid trans-eliminase (PATE), one of the enzymes involved in degradation of pectic substances. In a strain of Erwinia chrysanthemi, PATE is an extracellular enzyme and is almost totally excreted. In strains of Yersinia enterocolitica and Yersinia pseudotuberculosis, PATE is a periplasmic and cytoplasmic enzyme. In a strain of Erwinia carotovora, PATE activity is extracellular, periplasmic, and cytoplasmic. In an "oxytocum" strain of Klebsiella pneumoniae, PATE is entirely cytoplasmic; none is found in the periplasm and almost none in culture supernatants. Cells (but not supernatants, because PATE is not excreted) of K. pneumoniae show much higher levels of PATE activity when grown on polygalacturonate than when grown on other carbon sources. A clinical strain of Y. enterocolitica synthesizes high levels of PATE activity (almost all cell-bound) when grown in the presence of gluconate, glycerol, or polygalacturonate, and considerably less activity when grown on glucose. A clinical strain of Y. pseudotuberculosis synthesizes barely detectable quantities of cell-bound PATE regardless of the carbon source. The phytopathogenic Erwinia strains synthesize and excrete high levels of PATE activity when grown on polygalacturonate, galacturonate, gluconate, or glycerol. Growth on glucose represses PATE activity in the Erwinia strains; the extent of repression differs between the two strains. The different patterns of synthesis and excretion are discussed in the light of the possible catabolic and (or) cytolytic (plant tissue-macerating) functions and the related ecological significance of PATE in these enterobacteria.

Phenotypic suppression of a fructose-1,6-diphosphate aldolase mutation in Escherichia coli.

Strain NP 315 of Escherichia coli possesses a thermolabile fructose-1, 6-diphosphate (FDP) aldolase; its growth on carbohydrate substrates is inhibited probably as a consequence of the accumulation of high intracellular levels of FDP. Studies of one class of phenotypic revertants of strain NP 315 which have regained their ability to grow on C(6) substrates at 40 C showed that in these strains the buildup of the inhibitory FDP pool is prevented by additional mutations in enzymes catalyzing the conversion of the substrate offered in the medium to FDP. For example, mutations affecting 6-phosphogluconate dehydrogenase activity (gnd(-)) may be selected in great number without any mutagenesis and enrichment simply by isolating revertants of strain NP 315 able to grow on gluconate at 40 C. Similarly, an additional mutation in phosphoglucose isomerase (pgi(-)) restores the ability of these fda(-)gnd(-) strains to grow on glucose at 40 C. Glucose metabolism of these fda(-)gnd(-)pgi(-) strains was investigated. The enzymes of the Entner-Doudoroff pathway are induced to an appreciable extent upon growth of these mutants on glucose medium; further evidence for glucose degradation via this route (which normally is induced only in the presence of gluconate) was provided by following the fate of the C1 label of radioactive glucose in l-alanine. Predominant labeling of the carboxyl-carbon of l-alanine was observed, inciating a major contribution of the Entner-Doudoroff path to pyruvate formation from glucose. Chromatographic analysis of the intermediates of glucose metabolism showed further that glucose apparently is at least partly metabolized via a bypass consisting of the accumulation of extracellular gluconic acid which arises by dephosphorylation of 6-phosphogluconolactone and possibly of 6-phosphogluconate. This extracellular gluconate is then taken up and metabolized in the normal manner via the Entner-Doudoroff enzymes.

Xylitol Production by a Corynebacterium Species

The washed cells of a gluconate-utilizing Corynebacterium strain grown in a gluconatexylose medium produced xylitol from D-xylose in the presence of gluconate. The amount of xylitol was progressively increased with increasing gluconate concentration. An extract of cells grown in the gluconate-xylose medium showed NADPH-dependent D-xylose reductase activity and NADP-dependent 6-phosphogluconate dehydrogenase activity. These enzymes in the cell-free extract were purified by Sephadex G-100 gel filtration. The reduction of D-xylose to xylitol was demonstrated by the coupling the D-xylose reductase activity to the 6-phosphogluconate dehydrogenase activity with NADP as a cofactor using the cell-free extract and the fractionated enzymes.

Studies on the Uptake of Hexose Phosphates: I. 2-DEOXYGLUCOSE AND 2-DEOXYGLUCOSE 6-PHOSPHATE

Abstract 2-Deoxy-d-glucose was taken up by Escherichia coli and was recovered from cell extracts largely as a material that behaved chromatographically as 2-deoxyglucose 6-phosphate. With 14C-labeled deoxyglucose, it was observed that radioactivity was taken up by the cell and partially released after 1 hour. No induction of transport for glucose 6-phosphate was observed on treatment of cells with 2-deoxyglucose, in spite of the apparent accumulation of intracellular 2-deoxyglucose 6-phosphate. By contrast, extracellular 2-deoxyglucose 6-phosphate, in low concentration, was an effective inducer of the hexose phosphate transport system. 2-Deoxyglucose inhibited growth of E. coli and also caused temporary growth stasis when glycerol or succinate was the carbon source. Cells growing in the presence of gluconate or pyruvate resisted these effects of 2-deoxyglucose. When glucose-grown cells were forced to adapt to glycerol or succinate as carbon source in the presence of 2-deoxyglucose, the lag period was prolonged for as long as several days. Often this was followed by rapid growth caused by spontaneous appearance of a mutant strain. The mutant was isolated and found to be resistant to growth inhibition by deoxyglucose.

A New Method for Lysis of Blood Clots in vivo

Introduction According to current theory, blood clots are lysed in the body by a pro.teolytic enzyme, plasmin, which is produced from its precursor, plasminogen, by activators. The first activator was derived from streptococci by Tillet and Garner ( 1933 ) and named streptokinase; this bacterial metabolite is now assumed to transform a proactivator into an activator of plasminogen (Mullertz and Lassen, 195 3 ) . Various tissue activators have been recognized; of these the best known is urokinase, isolated by Williams (1951). Potent inhibitors in human blood normally prevent fibrinolysis. Presently two hypotheses of plasmin formation are advocated: (1) Plasmin and antiplasmin are bound in a complex, and fibrin competes with antiplasmin for plasmin; under certain conditions the plasmin-antiplasmin complex is disrupted and plasmin is released. (;2) When a fibrin clot forms, plasminogen is adsorbed on the surface of the fibrin molecules, later to be converted to plasmin by adhering activator, whereas in the circulating blood inhibitors prevent such activation. Isolation of the plasmin-antiplasmin comp!lex mentioned in the first hypothesis has not been reported; nor is there evidence of differential titers of plasminogen and plasmin in plasma prior to clotting and after lysis of the clot, which would support the second hypothesis. A fibrinolytic factor of bacterial origin, olperating independently of plasminogen, has been reported (Kopper, 1962). The factor dissolved untreated fibrin clots as well as fibrin clots heated at 85°C. for 45 minutes, a procedure which destroys plasminogen. The lytic factor was produced by the bacteria in the presence of gluconate from a dialyzable constituent of serum, or fibrin extract, or brain heart infusion. The purpose of the present study was to determine if the animal body, like the bacteria, possesses an enzyme capable of synthesizing a fibrinolysin in the presence of gluconate.