scholarly journals Effects of external pH on binding of external sulfate, 4.4-dinitro-stilbene-2,2'-disulfonate (DNDS), and chloride to the band 3 anion exchange protein.

1996 ◽  
Vol 107 (2) ◽  
pp. 293-306 ◽  
Author(s):  
S Q Liu ◽  
E Ries ◽  
P A Knauf
Keyword(s):  
Dissociation Constant ◽  
Competitive Inhibitor ◽  
Anion Exchange Protein ◽  
Or Groups ◽  
External Site ◽  
Selective Reagent ◽  
Guanidino Group ◽  
Transport Site ◽  
External Ph ◽  
Positively Charged

A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'-disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.

scholarly journals Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3.

1996 ◽  
Vol 107 (2) ◽  
pp. 271-291 ◽  
Author(s):  
S Q Liu ◽  
F Y Law ◽  
P A Knauf
Keyword(s):  
Rate Constant ◽  
Band 3 ◽  
Complex Model ◽  
Amino Acid Residues ◽  
Ping Pong ◽  
Extracellular Transport ◽  
Charged Form ◽  
Transport Site ◽  
External Ph ◽  
Positively Charged

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6-fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.

scholarly journals Transmembrane effects of intracellular chloride on the inhibitory potency of extracellular H2DIDS. Evidence for two conformations of the transport site of the human erythrocyte anion exchange protein.

1984 ◽  
Vol 83 (5) ◽  
pp. 657-681 ◽  
Author(s):  
W Furuya ◽  
T Tarshis ◽  
F Y Law ◽  
P A Knauf
Keyword(s):  
Anion Exchange ◽  
Protein Band ◽  
Competitive Inhibitor ◽  
Transport Protein ◽  
Inhibitory Potency ◽  
Intracellular Chloride ◽  
Anion Exchange Protein ◽  
Ping Pong ◽  
Chloride Exchange ◽  
Transport Site

The ping-pong model for the red cell anion exchange system postulates that the transport protein band 3 can exist in two different conformations, one in which the transport site faces the cytoplasm (Ei) and another in which it faces the outside medium (Eo). This model predicts that an increase in intracellular chloride should increase the fraction of sites in the outward-facing, unloaded form (Eo). Since external H2DIDS is a competitive inhibitor of chloride exchange that does not cross the membrane, it must bind only to the Eo form. Thus, an increase in Eo should cause an increase in H2DIDS inhibition. When intracellular chloride was increased at constant extracellular chloride, the inhibitory potency of H2DIDS rose, as predicted by the ping-pong model. This increase was not due to the concomitant changes in intracellular pH or membrane potential. When the chloride gradient was reversed, the inhibitory potency of H2DIDS decreased, again in qualitative agreement with the ping-pong model. These data provide support for the ping-pong model and also demonstrate that chloride gradients can be used to change the orientation of the transport protein.

scholarly journals Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine

1996 ◽  
Vol 317 (1) ◽  
pp. 267-272 ◽  
Author(s):  
Martin MEWIES ◽  
Leonard C. PACKMAN ◽  
F. Scott MATHEWS ◽  
Nigel S. SCRUTTON
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
Post Translational Modification ◽  
Mutant Proteins ◽  
Trimethylamine Dehydrogenase ◽  
Guanidino Group ◽  
Protein Environment ◽  
Mutagenic Changes ◽  
Positively Charged ◽  
Significant Mass

In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN. The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme. Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222. The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K). Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin. A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis. Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP. Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase. The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass. The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed.

Nature of taurodehydrocholic acid uptake in rat hepatocytes

1988 ◽  
Vol 254 (2) ◽  
pp. G269-G274 ◽  
Author(s):  
W. G. Hardison ◽  
P. J. Lowe ◽  
E. Gosink
Keyword(s):  
Membrane Potential ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Acid Uptake ◽  
Isolated Rat Hepatocytes ◽  
Acid Analogue ◽  
Sodium Gradient ◽  
Sodium Dependent ◽  
Independent Process ◽  
Transport Site

We studied uptake into isolated rat hepatocytes of the bile acid analogue taurodehydrocholate (TDHC) over a concentration range of 2.5-4,000 microM. Uptake was mainly by a saturable sodium-dependent process with a Km of approximately 50 microM and a Vmax of 0.036 nmol.s-1.mg protein-1. A lesser sodium-independent process was evident but was linear in the range studied. Both processes were inhibited by incubation at 37 degrees C under nitrogen in the presence of 3 mM sodium cyanide or by incubation at 0 degrees C. A single transport site was suggested by the Eadie-Hofstee plot of TDHC uptake from 2.5 to 750 microM. TDHC was a weak competitive inhibitor of taurocholic acid (TCA) uptake (Ki = 236 microM) but was not itself taken up by the TCA transport site. TCA exhibited moderately potent mixed inhibition of TDHC uptake. Uptake of both compounds was strongly inhibited by bromosulfophthalein (BSP) and Rose Bengal, whereas 0.5 mM alanine uptake was not affected. BSP exhibited a complex pattern of inhibition of TDHC uptake: mixed partial inhibition. Degree of inhibition of both TDHC and TCA uptake did not increase as BSP concentrations were increased from 50 to 100 microM. BSP did not exert its inhibitory effects by alteration of membrane potential or sodium gradients; 50 microM BSP changed membrane potential less than 10% and sodium gradient not at all. The data indicate that despite close structural analogy between TDHC and TCA, the two compounds are taken up by different sodium-dependent mechanisms. Nonetheless, the similar qualitative and quantitative effects of BSP on their uptakes suggests the mechanisms are related.

Ammonium transport by the colonic H+-K+-ATPase expressed in Xenopusoocytes

1999 ◽  
Vol 277 (2) ◽  
pp. C280-C287 ◽  
Author(s):  
Marc Cougnon ◽  
Patrice Bouyer ◽  
Frédéric Jaisser ◽  
Aleksander Edelman ◽  
Gabrielle Planelles
Keyword(s):  
Kinetic Analysis ◽  
Initial Rate ◽  
Functional Expression ◽  
Competitive Inhibitor ◽  
Ammonium Transport ◽  
Xenopus Laevis Oocytes ◽  
Intracellular Acidification ◽  
Extracellular Medium ◽  
Α Subunit ◽  
External Site

Functional expression of the rat colonic H+-K+-ATPase was obtained by coexpressing its catalytic α-subunit and the β1-subunit of the Na+-K+-ATPase in Xenopus laevis oocytes. We observed that, in oocytes expressing the rat colonic H+-K+-ATPase but not in control oocytes (expressing β1 alone), NH4Cl induced a decrease in86Rb uptake and the initial rate of intracellular acidification induced by extracellular NH4Cl was enhanced, consistent with [Formula: see text] influx via the colonic H+-K+-ATPase. In the absence of extracellular K+, only oocytes expressing the colonic H+-K+-ATPase were able to acidify an extracellular medium supplemented with NH4Cl. In the absence of extracellular K+ and in the presence of extracellular [Formula: see text], intracellular Na+ activity in oocytes expressing the colonic H+-K+-ATPase was lower than that in control oocytes. A kinetic analysis of86Rb uptake suggests that[Formula: see text] acts as a competitive inhibitor of the pump. Taken together, these results are consistent with[Formula: see text] competition for K+ on the external site of the colonic H+-K+-ATPase and with [Formula: see text] transport mediated by this pump.

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

1984 ◽  
Vol 83 (5) ◽  
pp. 683-701 ◽  
Author(s):  
P A Knauf ◽  
F Y Law ◽  
T Tarshis ◽  
W Furuya
Keyword(s):  
Anion Exchange ◽  
Chloride Transport ◽  
Chloride Concentration ◽  
Competitive Inhibitor ◽  
Disulfonic Acid ◽  
Inhibitory Potency ◽  
Intracellular Chloride ◽  
Chloride Exchange ◽  
A Site ◽  
Transport Site

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.

Proton inhibition of chloride exchange: asynchrony of band 3 proton and anion transport sites?

1986 ◽  
Vol 250 (6) ◽  
pp. C955-C969 ◽  
Author(s):  
M. A. Milanick ◽  
R. B. Gunn
Keyword(s):  
Conformational Changes ◽  
Chloride Concentration ◽  
Band 3 ◽  
Anion Transport ◽  
Band 3 Protein ◽  
Chloride Flux ◽  
Proton Binding ◽  
Chloride Exchange ◽  
Transport Site ◽  
External Ph

The inhibition of chloride exchange at 0 degrees C by protons at the cytoplasmic and the extracellular surface of the band 3 protein of human erythrocytes was measured between pH 4.6 and 7.6. At constant external pH and chloride concentration, internal protons were a mixed inhibitor of chloride flux, with the apparent pK2 = 6.1 for protonation of the inward-facing empty transporter conformation and the apparent pK3 = 5.7 for protonation of the chloride-transporter complex. The activation of chloride exchange by external chloride was inhibited by internal protons, and internal protonation of the externally facing empty conformation had a pK1 = 6.1. External protons were also a mixed inhibitor of chloride exchange with the apparent pK1 = 5.0 for the empty outward-facing transporter conformation. Because of the pHo dependence of self-inhibition, the value of pK3 on the outside for chloride could not be accurately determined, but the apparent pK3 for protonation of the iodide-transporter complex on the extracellular surface was 4.9. The data support a mechanism with a single proton binding site that can alternatively have access to the cytoplasmic and extracellular solutions. It appears that this proton binding and transport site can be coupled to the single anion transport site for cotransport, but the two sites can be on opposite sides of the membrane at the same time and thus can be asynchronously transported by conformational changes of band 3.

scholarly journals Utilization of Sodium Hexametaphosphate for Separating Scheelite from Calcite and Fluorite Using an Anionic–Nonionic Collector

Minerals ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 705 ◽  
Author(s):  
Jianhua Kang ◽  
Yuehua Hu ◽  
Wei Sun ◽  
Zhiyong Gao ◽  
Runqing Liu
Keyword(s):  
Photoelectron Spectroscopy ◽  
Negative Charge ◽  
Electrostatic Force ◽  
Sodium Hexametaphosphate ◽  
Infrared Spectrometry ◽  
Low Grade ◽  
X Ray ◽  
Charged Surfaces ◽  
Selective Reagent ◽  
Positively Charged

This study presents a highly selective reagent system that utilizes sodium hexametaphosphate (SHMP) to improve the separation of scheelite from calcite and fluorite using an anionic–nonionic collector. The recoveries of calcite and fluorite decreased to 20% as the SHMP dose exceeded 6 × 10−6 mol/L, whereas that of scheelite remained at 85%. The interaction mechanisms of minerals with SHMP were investigated through equilibrium speciation, Zeta potential, Fourier transform infrared spectrometry, and X-ray photoelectron spectroscopy analyses. SHMP exists as hydrogen phosphate anion in the aqueous solution with a pH of 7–12. Moreover, it may be adsorbed intensively on the positively charged surfaces of calcite and fluorite via electrostatic force or chelation with calcium ion to impede further adsorption of the assembled collector. By comparison, the adsorption of SHMP is feeble on the scheelite surface because of its negative charge. The roughing grade of low-grade scheelite ore is substantially improved from 0.74% to 1.65% compared with that in the contrast test in the absence of SHMP.

Covariation in regulation of affinity for branchial zinc and calcium uptake in freshwater rainbow trout.

1998 ◽  
Vol 201 (11) ◽  
pp. 1809-1815 ◽  
Author(s):  
C Hogstrand ◽  
N Webb ◽  
C M Wood
Keyword(s):  
Rainbow Trout ◽  
Fold Increase ◽  
Experimental Period ◽  
Competitive Inhibitor ◽  
Control Fish ◽  
Control Value ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Significant Linear Correlation ◽  
Ca Uptake ◽  
Transport Site

The possible coupling between regulation of the affinities for branchial Zn and Ca influx was investigated in juvenile rainbow trout Oncorhynchus mykiss acclimated to relatively hard fresh water ([Ca]=1.0 mmol l-1). The Km for branchial Ca influx was manipulated experimentally by exposing the fish to 2.3 micromol l-1 waterborne Zn for a total of 28 days. This procedure resulted in rapidly increased Km values for both Ca and Zn influx, an effect that remained through the experimental period. There was a significant linear correlation (r=0.88, P<0.02) between Km values for Ca and Zn measured at the same time points. Zn exposure caused progressively increasing maximum rate of transport, Jmax, values for Zn relative to the control value, but there was little, if any, effect on Jmax for Ca. These results support the idea of a shared transport site for Zn and Ca at the apical membrane of the gill epithelium and suggest that there is a certain degree of coregulation of branchial Zn and Ca uptake in rainbow trout. Removal of Ca from the water resulted in a large (six- to 24-fold) increase in affinity (decreased Km) for Zn influx and a modest (1.1- to 1.8-fold) increase in Jmax for Zn. Thus, Ca is a competitive inhibitor of Zn influx. In water lacking Ca, the Km for Zn in Zn-acclimated fish was no different from that of the control fish, suggesting that the Ca2+/Zn2+ transporter was regulated to improve Ca uptake.

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