Use of niflumic acid to determine the nature of the asymmetry of the human erythrocyte anion exchange system

Niflumic acid is a noncompetitive inhibitor of chloride exchange, which binds to a site different from the transport or modifier sites. When the internal Cl- concentration is raised, at constant extracellular Cl- , the inhibitory potency of niflumic acid increases. This effect cannot be attributed to changes in membrane potential, but rather it suggests that niflumic acid binds to the anion exchange protein band 3 only when the transport site faces outward. When the chloride gradient is reversed, with Clo greater than Cli , the inhibitory potency of niflumic acid decreases greatly, which indicates that the affinity of niflumic acid for band 3 with the transport site facing inward is almost 50 times less than when the transport site faces outward. Experiments in which Cli = Clo show no significant change in the inhibition by niflumic acid when Cl- is lowered from 150 to 10 mM. These data suggest that the intrinsic dissociation constants for Cl- at the two sides of the membrane are nearly equal. Thus, the chloride- loaded transport sites have an asymmetric orientation like that of the unloaded transport sites, with approximately 15 times more sites facing the inside than the outside. The asymmetry reflects an approximately 1.5 kcal/mol free energy difference between the inward-facing and outward-facing chloride-loaded forms of band 3. High concentrations of chloride (with Cli = Clo), which partially saturate the modifier site, have no effect on niflumic acid inhibition, which indicates that chloride binds equally well to the modifier site regardless of the orientation of the transport site.     

Regulation of Cl/HCO3 exchange in gastric parietal cells.

Microspectrofluorimetry of the fluorescent indicators 2',7'-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein and 6-methoxy-N-(3-sulfopropyl)-quinolinium was used to measure intracellular pH (pHi), intracellular Cl (Cli), and transmembrane fluxes of HCO3 and Cl in single parietal cells (PC) in isolated rabbit gastric glands incubated in HCO3/CO2-buffered solutions. Steady-state pHi was 7.2 in both resting (50 microM cimetidine) and stimulated (100 microM histamine) PCs. Transmembrane anion (HCO3 or Cl) flux rates during Cl removal from or readdition to the perfusate were the same in resting and stimulated PCs. These rates increased at alkaline pHi, though this pHi dependence was small in the physiological range. Maximum velocity (Vmax) for Cl influx or HCO3 efflux was 80-110 mM/min at pHi 7.6-7.8, and the Km for extracellular concentrations of Cl (Clo) was 25 mM; in the physiological range (pHi 7.1-7.3), Vmax for anion fluxes was approximately 50 mM/min. Steady-state Cli in the unstimulated PC was 62 +/- 5 mM, but on histamine stimulation, Cli decreased rapidly to 25 mM and then increased back to a steady-state level of 44 mM. HCO3 fluxes due to Cl removal or readdition were completely blocked by 0.5 mM 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS), but Cl fluxes were only inhibited by 80%. H2DIDS did not inhibit the decrease in Cli that occurred with histamine treatment. Diphenylamine carboxylate (0.5 mM) inhibited Cl flux by only 50% and caused no additional inhibition of Cl flux when used in conjunction with H2DIDS. Transmembrane anion fluxes during solution Cl removal or readdition occurred 80% through the anion exchanger at the basal membrane and 20% through other pathway(s), presumably the Cl channel in the apical membrane. We conclude that the increase in transport activity via the Cl/HCO3 exchanger that occurs during histamine-induced increases in HCl secretion is due mostly to the decrease in Cli. In the resting cell with Cli = 62 mM, Clo = 120 mM, pHi = 7.2, and extracellular pH = 7.4, the anion exchanger is poised near its thermodynamic equilibrium. During histamine stimulation Cli drops from 62 mM to 44 mM, the thermodynamic equilibrium of the anion exchanger at the basolateral membrane is disturbed, and the anion exchanger then exchanges cellular HCO3 for extracellular Cl. Cli serves a crucial regulatory role in stimulus-secretion coupling in the PC.     

Denaturation of a membrane transport protein by urea: The erythrocyte anion exchanger

Summary Chloride equilibrium exchange was measured in the presence of intracellular and extracellular urea, several different alkylureas and thiourea. Urea half-inhibited Cl exchange at about 2.5m, but the other, less polar analogs had significantly higher potencies; e.g., butylurea half-inhibited at about 60mm. Onset and reversal of inhibition occurred within less than 2 sec. The inhibition exhibited no obvious sigmoidal dependence on urea concentration, and at low concentrations dimethylurea was a noncompetitive inhibitor of Cl exchange. However, at higher concentrations the Dixon plots were curved upward and a Hill analysis of the dimethylurea data yielded a Hill coefficient of at least 1.5. When present on only one side of the membrane, the slowly penetrating thiourea inhibited Cl exchange with a higher potency from the outside of the cell. Cl/Br exchange was inhibited less under conditions of self-inhibition of anion exchange than in the absence of self-inhibition. These data indicate that the ureas inactivate the anion transporter by a reversible denaturation process, and that the function of the anion transport mechanism may be more sensitive to small perturbations of protein structure than are spectroscopically derived structural parameters.     

A new plot for the kinetic analysis of dead-end enzyme inhibitors

A new procedure to characterize reversible dead-end inhibitors is presented. Preliminary identification of the inhibitor type is made by plotting vo/vi against the inhibitor concentration at different substrate concentrations. The inhibition constants for competitive, uncompetitive and mixed dead-end inhibitors are determined by secondary plots of l/(slope) vs [S], l/(slope) vs l/[S] and (slope)(Ks + [S] vs [S] respectively. These secondary plots render straight lines only for their corresponding type of inhibitor. For noncompetitive inhibitors all the secondary plots used yield straight lines. Therefore, the application of this plotting procedure leads to unambiguous diagnosis of the inhibitor type. An important feature of the procedure presented here is that the variable used (vo/vi) is independent on Vmax values. Therefore, experimental values obtained from enzyme preparations showing significant differences in their specific activities -i.e. enzyme coming from different purification steps- can be used.     

Effects of the transport site conformation on the binding of external NAP-taurine to the human erythrocyte anion exchange system. Evidence for intrinsic asymmetry

External N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-taurine) inhibits human red cell chloride exchange by binding to a site that is distinct from the chloride transport site. Increases in the intracellular chloride concentration (at constant external chloride) cause an increase in the inhibitory potency of external NAP-taurine. This effect is not due to the changes in pH or membrane potential that usually accompany a chloride gradient, since even when these changes are reversed or eliminated the inhibitory potency remains high. According to the ping-pong model for anion exchange, such transmembrane effects of intracellular chloride on external NAP-taurine can be explained if NAP-taurine only binds to its site when the transport site is in the outward-facing (Eo or EClo ) form. Since NAP-taurine prevents the conformational change from EClo to ECli , it must lock the system in the outward-facing form. NAP-taurine can therefore be used just like the competitive inhibitor H2DIDS (4,4'-diisothiocyano-1,2- diphenylethane -2,2'-disulfonic acid) to monitor the fraction of transport sites that face outward. A quantitative analysis of the effects of chloride gradients on the inhibitory potency of NAP-taurine and H2DIDS reveals that the transport system is intrinsically asymmetric, such that when Cli = Clo, most of the unloaded transport sites face the cytoplasmic side of the membrane.     

Relative contributions of the slippage and tunneling mechanisms to anion net efflux from human erythrocytes

The rates of anion net efflux from gramicidin-treated erythrocytes in the presence of a K gradient were measured at 25 degrees C, pH 7.8, as rates of loss of Ki. The experiments served to estimate the relative contributions of two hypothetical mechanisms to Cl net efflux at low extracellular Cl concentrations. Cl, Br, and NO3 net effluxes were measured into media of different Cl, Br, or NO3 concentrations, respectively, to determine and compare the relative rates of the extracellular anion-inhibitable components. They were 48, 160, and 230 mmol/(kg Hb X min), respectively, at a membrane potential of about -90 mV. This indicates that the anion-inhibitable efflux is not due solely to the return translocation of the empty transport site ("slippage") because slippage should be independent of the chemical nature of the anion. Cl net efflux was also measured as a function of the intracellular Cl concentration into media containing either 0 or 50 mM Cl. Under both conditions, net efflux was linearly dependent on Cli between 30 and 300 mM Cli and was 0 when back-extrapolated to 0 Cli. This observation is not compatible with the slippage process, which under these conditions would have been expected to be independent of Cli above 15 mM Cli. It was concluded that slippage contributes negligibly to Cl net efflux even at low extracellular anion concentrations and that the alternative process of "tunneling"--that is, movement of the anion through the anion transporter without a conformational change in a channel-type behavior--is the major, if not the sole, mechanism underlying Cl conductance.     

Steady-state kinetic mechanism of Ras farnesyl:protein transferase.

The steady-state kinetic mechanism of bovine brain farnesyl:protein transferase (FPTase) has been determined using a series of initial velocity studies, including both dead-end substrate and product inhibitor experiments. Reciprocal plots of the initial velocity data intersected on the 1/[s] axis, indicating that a ternary complex forms (sequential mechanism) and suggesting that the binding of one substrate does not affect the binding of the other. The order of substrate addition was probed by determining the patterns of dead-end substrate and product inhibition. Two nonhydrolyzable analogues of farnesyl diphosphate, (alpha-hydroxyfarnesyl)phosphonic acid (1) and [[(farnesylmethyl)hydroxyphosphinyl]methyl]phosphonic acid (2), were both shown to be competitive inhibitors of farnesyl diphosphate and noncompetitive inhibitors of Ras-CVLS. Four nonsubstrate tetrapeptides, CV[D-L]S, CVLS-NH2, N-acetyl-L-penicillamine-VIM, and CIFM, were all shown to be noncompetitive inhibitors of farnesyl diphosphate and competitive inhibitors of Ras-CVLS. These data are consistent with random order of substrate addition. Product inhibition patterns corroborated the results found with the dead-end substrate inhibitors. We conclude that bovine brain FPTase proceeds through a random order sequential mechanism. Determination of steady-state parameters for several physiological Ras-CaaX variants showed that amino acid changes affected the values of KM, but not those of kcat, suggesting that the catalytic efficiencies (kcat/KM) of Ras-CaaX substrates depend largely upon their relative binding affinity for FPTase.     

Kinetic mechanism of beef pancreatic L-asparagine synthetase

The kinetic mechanism of bovine pancreatic asparagine synthetase was deduced from initial velocity studies and product inhibition studies of both the glutamine-dependent and ammonia-dependent reactions. For the glutamine-dependent pathway, parallel lines were observed in the double reciprocal plot of 1/V vs. 1/[glutamine] at varied aspartate concentrations, and in the plot of 1/V vs. 1/[ATP] at varied aspartate concentrations. Intersecting lines were found for the plot of 1/V vs. 1/[ATP] at varied glutamine concentrations. Product inhibition patterns, including dual inhibitor studies for measuring the synergistic effects of multiproduct inhibition, were used to support an ordered bi-uni-uni-ter ping-pong mechanism. Glutamine and ATP sequentially bind, followed by the release of glutamate and the addition of aspartate. Pyrophosphate, AMP, and asparagine are then sequentially released. When the ammonia-dependent reaction was studied, it was found that the mechanism was significantly different. NH3 bound first followed by a random addition of ATP and aspartate. Pyrophosphate, AMP, and asparagine were then sequentially released as in the glutamine-utilizing mechanism. From these studies, a comprehensive mechanism has been proposed through which either glutamine or NH3 can provide nitrogen for asparagine production from aspartate.     

Chloride net efflux from intact erythrocytes under slippage conditions. Evidence for a positive charge on the anion binding/transport site

Tracer chloride and potassium net efflux from valinomycin-treated human erythrocytes were measured into media of different chloride concentrations, Clo, at 25 degrees C and pH 7.8. Net efflux was maximal [45-50 mmol (kg cell solids)-1 min-1] at Clo = 0. It decreased hyperbolically with increasing Clo to 14-16 mmol (kg cell solids)-1 min- 1. Half-maximal inhibition occurred at Clo = 3 mM. In the presence of the anion exchange inhibitor DNDS, net efflux was reduced to 5 mmol (kg cell solids)-1 min-1, independent of Clo. Of the three phenomenological components of net efflux, the Clo-inhibitable (DNDS-inhibitable) component was tentatively attributed to "slippage," that is, net transport mediated by the occasional return of the empty transporter. The Clo-independent (DNDS-inhibitable) component was tentatively attributed to movement of chloride through the anion transporter without the usual conformational change of the transport site on the protein ("tunneling"). These concepts of slippage and tunneling are shown to be compatible with a model that describes the anion transporter as a specialized single-site, two-barrier channel that can undergo conformational changes between two states. Net chloride efflux when the slippage component dominated (Clo = 0.7 mM) was accelerated by a more negative (inside) membrane potential. It appears that the single anion binding-and-transport site on each transporter has one net positive charge and that is neutralized when a chloride ion is bound.     

Activity of gamma-glutamyl transferase of sheep cerebral cortex with respect to amino acids and glutathione]

gamma-glutamyl Transferase fron Sheep brain cortex capillaries was studied from the point of view of transport of aminoacids across blood brain barrier. Excess substrate inhibition was competitive and observed both with donor (glutathione) and various acceptors (methionine, alanine, tryptophan) but not with arginine. Excess glutathione inhibition of transfer reaction is concomitant with an increase of total reaction (transfer + hydrolysis + autotranspeptidation). With regard to aminoacids, the greater the K'm the stronger the inhibition. This inhibition is the result of formation of a dead complex. Lineweaver-Burk plots 1/v versus 1/[acceptor] give straight lines meeting at the same point, whereas 1/v verus 1/[donor] plots are roughly parallel for high aminoacid concentrations and become secant for the low ones. Replots of slopes vs. 1/[acceptor] are not linear: the lower the aminoacid affinity the more pronounced the slope replot curvature. Thus kinetic patterns are consistent with a branched ping-pong mechanism including a ternary complex (Enzyme-acceptor-H2O) at high or low relative concentration, which balances the two branches. The estimated value of kinetic parameters does not support the hypothesis of major implication of the enzyme in brain uptake of aminoacids.     

Further studies of the volume-regulatory response of Amphiuma red cells in hypertonic media. Evidence for amiloride-sensitive Na/H exchange

When Amphiuma red cells are shrunken in hypertonic media, they return toward their original volume by gaining Na through an amiloride-sensitive pathway. As cells recover their volume during this volume-regulatory increase (VRI) response, acid is extruded into the medium. Medium acidification is correlated with cell Na uptake. Both medium acidification and cell Na uptake are blocked by 10(-3) M amiloride or by replacing medium Na with K or choline. Perturbations that increase cell Na uptake (such as increasing medium osmolality) also increase medium acidification. As the medium becomes more acidic, the cells become more alkaline. These changes in cell and medium pH are increased if pH equilibration across the cell membrane is prevented by inhibiting the anion exchanger with SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid). The quantity of acid extruded by SITS-treated cells is the same as the quantity of Na gained, which strongly suggests 1:1 exchange of Na for H. Cell enlargement in SITS-treated cells results from the exchange of osmotically active Na ions for H ions that are not osmotically active when combined with cellular buffers. Previous evidence indicates that the normal VRI response involves an increase in the cellular content of Cl as well as Na. We show that SITS completely blocks net Cl uptake, which suggests that Cl enters via the anion exchanger. SITS also slows Na entry, presumably as a result of the above-mentioned increase in cell pH caused by SITS. We suggest that the initial event in the VRI response is net Na uptake via a Na/H exchanger, and that net Cl uptake results from secondary Cl/HCO3 exchange via the anion exchanger.     

The human erythrocyte anion transport protein, band 3. Characterization of exofacial alkaline titratable groups involved in anion binding/translocation

Chloride self-exchange across the human erythrocyte membrane at alkaline extracellular pH (pHO) and constant neutral intracellular pH (pH(i)) can be described by an exofacial deprotonatable reciprocating anion binding site model. The conversion of the transport system from the neutral to the alkaline state is related to deprotonation of a positively charged ionic strength- and substrate-sensitive group. In the absence of substrate ions ([ClO] = 0) the group has a pK of approximately 9.4 at constant high ionic strength (equivalent to approximately 150 mM KCl) and a pK of approximately 8.7 at approximately zero ionic strength. The alkaline ping-pong system (examined at constant high ionic strength) demonstrates outward recruitment of the binding sites with an asymmetry factor of approximately 0.2, as compared with the inward recruitment of the transport system at neutral pHO with an asymmetry factor of approximately 10. The intrinsic half-saturation constant for chloride binding, with [Cli] = [Clo], increased from approximately 30 mM at neutral to approximately 110 mM at alkaline pHO. The maximal transport rate was a factor of approximately 1.7 higher at alkaline pHO. This increase explains the stimulation of anion transport, the "modifier hump," observed at alkaline pHO. The translocation of anions at alkaline pHO was inhibited by deprotonation of another substrate-sensitive group with an intrinsic pK of approximately 11.3. This group together with the group with a pK of approximately 9.4 appear to form the essential part of the exofacial anion binding site. The effect of extracellular iodide inhibition on chloride transport as a function of pHO could, moreover, be simulated if three extracellular iodide binding constants were included in the model: namely, a competitive intrinsic iodide binding constant of approximately 1 mM in the neutral state, a self-inhibitor binding constant of approximately 120 mM in the neutral state, and a competitive intrinsic binding constant of approximately 38 mM in the alkaline state.     

Liquid and solid-state Cl- -sensitive microelectrodes. Characteristics and application to intracellular Cl- activity in Balanus photoreceptor

When intracellular chloride activity (aiCl) was monitored with chloride-sensitive liquid ion exchanges (CLIX) microelectrodes in Balanus photoreceptors, replacement of extracellular chloride (Cl0) by methanesulfonate or glutamate was followed by a rapid but incomplete loss of aiCl. When propionate was used as the extracellular anion substitute, CLIX electrodes detected an apparent gain in aiCl, while a newly designed Ag-AgCl wire-in glass microelectrode showed a loss of aiCl under the same conditions. This discrepancy in Cl- washout when propionate replaced Cl0 is explained by the differences in selectivity of CLIX and Ag-AgCl electrodes for native intracellular anions and for the extracellular anion substitute which also replaces Cli and interferes in the determination of aiCl. Both electrodes indicate that ECl approximately Em when the cells are bathed in normal barnacle saline, and both electrodes showed the rate of Cl washout (tau approximately 5 min) to be independent of Cli when Cl0 was replaced by glutamate. Details of Ag-AgCl microelectrode construction are presented. These electrodes were tested and found to be insensitive to the organic anion substitutes used in this study. Selectivity data of CLIX electrodes for several anions of biological interest are described.     

Steady-state kinetic analysis for the reaction of ammonium and alkylammonium ions with methylamine dehydrogenase from bacterium W3A1

The steady-state kinetic mechanism for the reaction of n-alkylamines and phenazine ethosulfate (PES) or phenazine methosulfate (PMS) with methylamine dehydrogenase from bacterium W3A1 is found to be of the ping-pong type. This conclusion is based on the observations that 1/v versus 1/[methylamine] or 1/[butylamine] plots, at various constant concentrations of an oxidizing substrate, and 1/v versus 1/[PES] or 1/[PMS] plots, at various constant concentrations of a reducing substrate, are parallel. Additionally, the values of kcat/Km for four n-alkylamines are identical when PES is the oxidizing substrate, as were the kcat/Km values for four reoxidizing substrates when methylamine was the reducing substrate. Last, analysis of steady-state kinetic data obtained when methylamine and propylamine are presented to the enzyme simultaneously and PES and PMS are used simultaneously also supports the involvement of a ping-pong mechanism. The enzymic reaction with either methylamine or PES is dependent on the ionic strength, and the data indicate that each interacts with an anionic site on methylamine dehydrogenase. The presence of ammonium ion at low concentration activates the enzyme, but at high concentration this ion is a competitive inhibitor in the reaction involving methylamine and the enzyme. A complete steady-state mechanism describing these ammonia effects is presented and is discussed in light of the nature of the pyrroloquinoline quinone cofactor covalently bound to the enzyme.     

Evidence that anion transport by band 3 proceeds via a ping-pong mechanism involving a single transport site. A 35 Cl NMR study

Band 3 catalyzes the one-for-one exchange of monovalent anions across the red cell membrane. At least two anion binding sites have been postulated to exist on the transport unit: 1) a transport site that has been observed by saturation kinetics and by 35 Cl NMR studies of chloride binding, and 2) a 35Cl NMR-invisible inhibitory site that has been proposed to explain the inhibition of anion exchange at large anion concentrations. A number of independent studies have indicated that the transport site is alternately exposed to different sides of the membrane during the transport cycle. Yet the role, if any, of the postulated inhibitory site in the transport cycle is not known. Here it is shown that: 1) when the [Cl-], [Br-], or pH is varied, the band 3 transport sites on both sides of the membrane behave like a homogeneous population of simple anion binding sites in 35Cl NMR experiments, and 2) when the [Cl-] is varied, the outward-facing transport site behaves like a simple anion binding site. These results indicate that the postulated inhibitory site has no effect on chloride binding to the transport site. Instead, the results are quantitatively consistent with the ping-pong model (Gunn, R. B., and Fr?lich, O. (1979) J. Gen. Physiol. 74, 351-374), which states that the transport site is the only site involved in the transport cycle. Expressions are derived for the macroscopically observed characteristics of a ping-pong transporter: these characteristics are shown to be weighted averages of the microscopic properties of the inward- and outward-facing conformations of the transport site. In addition to supporting the simplicity of the transport mechanism, the high pH titration curve for chloride binding to the transport site provides insight into the structure of the site. The macroscopically observed pKA = 11.1 +/- 0.1 in the leaky ghost system indicates that an arginine must provide the essential positive charge in the inward- or outward-facing conformation of the transport site, or in both conformations.     

Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic squalamine.

Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 microM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 microM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.     

Enhanced formation of a HCO3- transport metabolon in exocrine cells of Nhe1-/- mice

Cl(-) influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked Cl(-)/HCO(3)(-) (Ae) and Na(+)/H(+) (Nhe) exchange mechanisms. The dependence of this major Cl(-) uptake pathway on Na(+)/H(+) exchanger expression was examined in the parotid acinar cells of Nhe1(-/-) and Nhe2(-/-) mice, both of which exhibited impaired fluid secretion. No change in Cl(-)/HCO(3)(-) exchanger activity was detected in Nhe2-deficient mice. Conversely, Cl(-)/HCO(3)(-) exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in Cl(-)/HCO(3)(-) exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1(-/-) mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced Cl(-)/HCO(3)(-) exchange activity was accompanied by an increased abundance of Car2.Ae2 complexes in the parotid plasma membranes of Nhe1(-/-) mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2.Ae2 HCO(3)(-) transport metabolon in parotid exocrine cell function. Increased abundance of this HCO(3)(-) transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1(-/-) mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.     

Studies on the sucrose phosphate synthetase kinetic mechanism

The kinetic properties of wheat germ sucrose phosphate synthetase, which catalyzes the reaction UDP-glucose + fructose 6-phosphate → UDP + sucrose 6-phosphate have been studied. A plot of the reciprocal initial velocity versus reciprocal substrate concentration gave a series of intersecting lines indicating a sequential mechanism. Product inhibition studies showed that UDP was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. A dead-end inhibitor, inorganic phosphate, was competitive with UDP-glucose and noncompetitive with fructose 6-phosphate. The results of initial velocity and product and dead-end inhibition studies suggested that the addition of substrates to the enzyme follows an ordered mechanism.     

Acetylcholinesterase. I. Kinetic aspects of interaction with reversible effectors]

Interaction of an effector M with acetylcholinesterase (EC 3.1.1.7) according to the model of Krupka and Laidler was analysed. Some usual functions of [M] : 1/VM, [(VO/VM)-1]/[M] (where VO and VM are the steady state rates in the absence and in the presence of modifiers, respectively), vertical intercept 1/VM, slope KM/VM and absolute value of reciprocal horizontal intercept KM of Lineweaver-Burk plots are investigated and corresponding plots described. It is particularly shown that if Dixon plots are curves concave downwards, plots of [VO/VM)-1]/[M] and 1/VM against [M] are hyperbolas concave upwards and downwards respectively. If Dixon plots are curves concave upwards, plots of [(VO/VM)-1]/[M] and 1/VM versus [M] are hyperbolas concave downwards and upwards respectively. Moreover plots of KM/VM against [M] are linear. However, this model does not explain some observations, under conditions of high ionic strength (gamma/2 greater than or equal to 0,1), where Dixon plots are curves concave upwards, plots of [VO/VM)-1]/[M] versus [M] strainght lines, the plot of 1/VM against [M] is a straight line or a curve concave upwards of positives slopes and the plot of KM/VM versus [M] a curve of positive slope concave upwards. These experimental data might be interpreted by an extension of the preceding model to a mechanism with two enzymatic binding sites under kinetic conditions that are determined.     

Reaction of neuronal nitric-oxide synthase with 2,6-dichloroindolphenol and cytochrome c3+: influence of the electron acceptor and binding of Ca2+-activated calmodulin on the kinetic mechanism

Binding of Ca(2+)-activated calmodulin (Ca(2+)-CaM) to neuronal nitric-oxide synthase (nNOS) increases the rate of 2,6-dichloroindolphenol (DCIP) reduction 2-3-fold and that of cytochrome c(3+) 10-20-fold. Parallel initial velocity patterns indicated that both substrates were reduced via two-half reactions in a ping-pong mechanism. Product and dead-end inhibition data with DCIP were consistent with an iso ping-pong bi-bi mechanism; however, product and dead-end inhibition studies with cytochrome c(3+) were consistent with the (two-site) ping-pong mechanism previously described for the NADPH-cytochrome P450 reductase-catalyzed reduction of cytochrome c(3+) [Sem, D., and Kasper, C. (1994) Biochemistry 33, 12012--12021]. Dead-end inhibition by 2'-adenosine monophosphate (2'AMP) was competitive versus NADPH for both electron acceptors, although the value of the slope inhibition constant, K(is), was 25-30-fold greater with DCIP as the substrate than with cytochrome c(3+). The difference in the apparent affinity of 2'AMP is proposed to result from a rapidly equilibrating isomerization step that occurs in both mechanisms prior to the binding of NADPH. Thus, initial velocity, product, and dead-end inhibition data were consistent with a di-iso ping-pong bi-bi and an iso (two-site) ping-pong mechanism for the reduction of DCIP and cytochrome c(3+), respectively. The presence Ca(2+)-CaM did not alter the proposed kinetic mechanisms. The activated cofactor had a negligible effect on (k(cat)/K(m))(NADPH), while it increased (k(cat)/K(m))(DCIP) and (k(cat)/K(m))(cytc) 4.5- and 23-fold, respectively.