Spatial requirements for insulin-sensitive sugar transport in rat adipocytes

(1) Alkyl sugar inhibition of d-allose uptake into adipocytes has been used to explore the spatial requirements of the external sugar transport site in insulin-treated cells. α-methyl and β-methyl glucosides show low affinity indicating very little space around C-1. The high affinity of d-glucosamine (K_i = 9.05 ± 0.66 mM) is lost by N-acetylation. $\text{N-}\text{Acetyl-}\text{d}\text{-glucosamine}$ shows no detectable affinity, indicating that a bulky group at C-2 is not accepted. Similarly $\text{2,3-}\text{di}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ (K_i = 42.1 ± 7.5 mM) has lower affinity than $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$ (K_i = 5.14 ± 0.32 mM) indicating very little space around C-2 but much more around C-3. A reduction in affinity does occur if a propyl group is introduced into the C-3 position. The K_i for $\text{3-O-}\text{propyl-}\text{d}\text{-glucose}$ is 11.26 ± 2.12 mM. $\text{6-O-}\text{Methyl-}\text{d}\text{-galactose}$ (K_i = 87.2 ± 17.9 mM) and $\text{6-O-}\text{propyl-}\text{d}\text{-glucose}$ (K_i = 78.07 ± 12.6 mM) show low affinity compared with d-galactose and d-glucose, indicating steric constraints around C-6. High affinity is restored in $\text{6-O-}\text{pentyl-}\text{d}\text{-galactose}$ (K_i = 4.66 ± 0.23 mM) possibly indicating a hydrophobic binding site around C-6). (2) In insulin treated cells $\text{4,6-O-}\text{ethylidene-}\text{d}\text{-glucose}$ (K_i = 6.11 ± 0.5 mM) and maltose (K_i = 23.5 ± 2.1 mM) are well accommodated by the site but trehalose shows no detectable inhibition. These results indicate that the site requires a specific orientation of the sugar as it approaches the transporter from the external solution. C-1 faces the inside while C-4 faces the external solution. (3) To determine the spatial and hydrogen bonding requirements for basal cells 40 μM $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$ was used as the substrate. Poor hydrogen bonding analogues and analogues with sterically hindering alkyl groups showed similar K_i values to those determined for insulin-treated cells. These results indicate that insulin does not change the specificity of the adipocyte transport system.     

4-Azidophlorizin,a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative ($\text{2′-O-(β-}\text{d}\text{-}\text{glucopyranosyl}\text{)-4-}\text{azidophloretin}$, 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na⁺ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′_i, the constant for fully-competitive inhibition of (Na⁺, Δψ)-dependent d-glucose uptake, (2) K′_d, the dissociation constant of 4-azido[³H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″_i, the constant for fully-competitive inhibition of the specific ((Na⁺, Δψ)-dependent, d-glucose protectable) high-affinity [³H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (K′_i, K′_d and K″_i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′_i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa

A kinetic study on esterification between d-glucose and l-phenylalanine catalysed by lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) in organic media investigated in detail showed that both the lipases followed a Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical double reciprocal plots and computer simulation studies showed that competitive double substrate inhibition took place at higher concentrations leading to dead-end inhibition in the case of RML and in the case of CRL, inhibition only by d-glucose at higher concentrations leading to dead-end lipase–d-glucose complexes. An attempt to obtain the best fit of these kinetic models through curve-fitting yielded in good approximation, the apparent values of important kinetic parameters, RML: k _cat = 2.24 ± 0.23 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 95.6 ± 9.7 mM, K _{m d-glucose} = 80.0 ± 8.5 mM, K _{i l-phenylalanine} = 90.0 ± 9.2 mM, K _{i d-glucose} = 13.6 ± 1.42 mM; CRL: k _cat = 0.51 ± 0.06 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 10.0 ± 0.98 mM, K _{m d-glucose} = 6.0 ± 0.64 mM, K _{i d-glucose} = 8.5 ± 0.81 mM.     

Mechanism of the sarcoplasmic reticulum calcium pump. Fluorometric study of the phosphorylated intermediates.

After removal of calcium ions bound to the high affinity sites the sarcoplasmic reticulum calcium pump can be phosphorylated by inorganic phosphate. The intrinsic fluorescence of the protein is used to follow conformational changes of the pump and an intensity change can be observed upon addition of phosphate. This effect is activated by internal calcium ($\text{K}\text{1}\text{2}\text{= 10 mM}$) and inhibited by external calcium ($\text{K}\text{1}\text{2}\text{= 0.4 μM}$) and the apparent affinity for phosphate is high (0.2 mM). We conclude that the change observed is linked to the formation of the gradient-dependent phosphorylated intermediate. It is compared with previous results concerning the enzymatic cycle of the pump.     

Effect of internal and external K+ on Na+−Ca2+ exchange in dialyzed squid axons under voltage clamp conditions

The effect of external and internal K⁺ on Na_o⁺-dependent Ca²⁺ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K⁺ (up to 70 mM) has no effect on Na⁺?Ca²⁺ exchange. Removal of K_i⁺ causes a marked inhibition in the Na_o⁺-dependent Ca²⁺ efflux component. Internal K⁺ activates the Na⁺?Ca²⁺ exchange with low affinity $\text{(K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 90 mM)}$. Activation by K_i⁺ is similar in the presence or in the absence of Na_i⁺, thus ruling out a displacement of Na_i⁺ from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca²⁺ efflux on K_i⁺. The present results demonstrate that K_i⁺ is an important cofactor (partially required) for the proper functioning of the forward Na⁺?Ca²⁺ exchange.     

Characterization of the (Na+ + K+)-ATPase in a membranous preparation from the optic ganglion of the squid (Loligo pealei)

(1) A membrane fraction enriched in $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ specific activity of approx. 2 units/mg and was $\text{>95\%}$ ouabain-sensitive. (2) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ had a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of $\text{0.42 ± 0.04}\text{mM}$ and a pH optimum of 7.0. It was inhibited by ouabain with a $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ of $\text{0.32 ± 0.04 μ}\text{M}$. (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with $\text{NaCl + KCl}\text{= 300}\text{mM}$. (4) The Mg²⁺ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the$\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Mg²⁺ was $\text{0.86 ± 0.10}\text{mM}$, and at 6 mM ATP, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ was $\text{1.86 ± 0.44}\text{mM}$. High levels of Mg²⁺ caused inhibition of hydrolysis. (5) The interactions of Na⁺ and K⁺ were examined over a range of conditions. K⁺ levels caused modulations in the Na⁺ dependence in the range of 1–150 mM. (6) The $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves.     

Inhibition by dexamethasone of the in vitro transport of 3-O-methylglucose into rat thymocytes

Reduced glucose transport across the plasma membrane and reduced phosphorylation may both be responsible for the early inhibitory effect of physiological concentrations of glucocorticoids on glucose uptake by rat thymocytes.The early inhibitory effects of glucocorticoids (5 · 10^?7 M dexamethasone) on glucose consumption and ¹⁴CO₂ formation from d-[U-¹⁴C]glucose were reproduced.The total uptake curve of 4.8 μM $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyl-}\text{d}\text{-glucose}$ was biexponential with $\text{t}\text{1}\text{2}$ of 1.1 min and 36 min, respectively, the rapid part comprising about 50% of the equilibrated intracellular water space. The latency of the effect of 5 · 10^?7 M dexamethasone on $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyl-}\text{d}\text{-glucose}$ uptake ranged from 15 to 100 min and the inhibition varied from 15 to 55% independently of the lag period. The effect of 3-O-methylglucose concentration on the initial uptake by steroid-responsive cell preparations was tested after 45 min of preincubation with or without 5 · 10^?7 M dexamethasone. In 12 experiments dexamethasone reduced V from 1.36 ± 0.16 mmol · min^?1 · l^?1 cell water to 0.81 ± 0.10 mmol · min^?1 · l^?1 cell water with insignificant change of K_m (6.0 mM versus 5.9 mM). Dexamethasone had similar effect after 90 or 120 min.The variabilities of control cell transport capacity, the lag period and the magnitude of the dexamethasone effect could not be accounted for by changes in pH, effects of cell density, concentrations of albumin, ethanol, nucleosides, pyruvate or correlated to age and sex of the rats. In conclusion the inhibition of glucocorticoids on glucose consumption by thymocytes appears to be an inhibited plasma membrane transport capacity.     

Steady-state kinetics of ADP-arsenate and ATP-synthesis in Rhodospirillum rubrum chromatophores

(1) Rates of ATP synthesis and ADP-arsenate synthesis catalyzed by Rhodospirillum rubrum chromatophores were determined with the firefly luciferase method and by a coupled enzyme assay involving hexokinase and glucose-6-phosphate dehydrogenase. (2) V_m for ADP-arsenate synthesis was about 2-times lower than V_m for ATP-synthesis. With saturating [ADP], K(As_i) was about 20% higher than K(P_i). With saturating [anion], K(ADP) was during arsenylation about 20% lower than during phosphorylation. (3) Plots of $\text{1}\text{v}$ vs. $\text{1}\text{[}\text{substrate}\text{]}$ were non-linear at low concentrations of the fixed substrate. The non-linearity was such as to suggest a positive cooperativity between sites binding the variable substrate, resulting in an increased $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ratio. High concentrations of the fixed substrate cause a similar increase in $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext>}}$, but abolish the cooperativity of the sites binding the variable substrate. (4) Low concentrations of inorganic arsenate (As_i) stimulate ATP synthesis supported by low concentrations of P_i and ADP about 2-fold. (5) At high ADP concentrations, the apparent K_i of As_i for inhibition of ATP-synthesis was 2–3-times higher than the apparent K_m of As_i for arsenylation; the apparent K_i of P_i for inhibition of ADP-arsenate synthesis was about 40% lower than the apparent K_m of P_i for ATP synthesis. (6) The results are discussed in terms of a model in which P_i and As_i compete for binding to a catalytic as well as an allosteric site. The interaction between these sites is modulated by the ADP concentration. At high ADP concentrations, interaction between these sites occurs only when they are occupied with different species of anion.     

Inhibition of anion transport in human red blood cells by 5,5′-dithiobis(2-nitrobenzoic acid)

The uptake of [³²P]phosphate into human red blood cells was inhibited ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 0.6}\text{mM}$) by the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). 2-Nitro-5-thiobenzoic acid (NTB), the reduced form of DTNB, was a less potent inhibitor ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7}\text{mM}$). The inhibition of anion transport by DTNB could be reversed by washing DTNB-treated cells with isotonic buffer, or by incubating DTNB-treated cells with 2-mercaptoethanol, which converted DTNB to NTB. DTNB competitively inhibited the binding of 4-[¹⁴C]-benzamido-4′-aminostilbene-2,2′-disulfonate, a potent inhibitor of anion transport ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 1?2 μ}\text{M}$), to band 3 protein in cells and ghost membranes. These results suggest that the stilbene-disulfonate binding site in band 3 protein can readily accommodate the organic anion DTNB, and that inhibition by DTNB was not due to reaction with an essential sulfhydryl group.     

Glutamate transport in pig heart mitochondria. Binding and structural properties of high-glutamate affinity proteolipid: Reconstitution studies

Previously, a proteolipid that can bind glutamate with high affinity has been isolated from pig heart mitochondrial membranes. A final affinity chromatography on γ-methylglutamate-albumin coreticulated on glass fiber was necessary. This procedure includes long dialysis steps which tend to denature the high-glutamate affinity proteolipid.Here is described a new method of isolation which avoids long dialysis steps and yields greater amounts of the high-glutamate affinity proteolipid.The binding of glutamate or aspartate on high-glutamate affinity proteolipid has been studied by gel filtration, by equilibrium dialysis or by a new procedure of rapid centrifugation based on the insolubility of high-glutamate affinity proteolipid in water. The latter method permits the detection of low and high affinity sites for glutamate with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ 60 mM and 55 μM, respectively. Among a series of analogues, aspartate appeared to be the best competitor: $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 30 μ}\text{M}$ and two $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values, 0.37 mM (at high glutamate concentration) and 3.8 μM (at low glutamate concentration). High-glutamate affinity proteolipid binds 0.4 nmol of glutamate but only 0.1 nmol of aspartate per mg protein. The sites for glutamate and aspartate appear to be different but interdependent.In the presence of high-glutamate affinity proteolipid, externally added glutamate stimulated the efflux of aspartate from preloaded liposomes.High-glutamate affinity proteolipid contains cardiolipin, phosphatidyl choline and phosphatidyl ethanolamine the distribution of which is different from that of the inner membrane.The effects of various phospholipases, trypsin, and thiol reagents were studied on the binding of glutamate. High-glutamate affinity proteolipid binds 9 nmol $\text{N-}\text{ethylmaleimide}$ per mg protein but only 6.1 nmol in the presence of glutamate. The dissociation of high-glutamate affinity proteolipid caused by thiol reagents yielded a soluble protein fraction with higher affinity for glutamate.Electrophoresis and an immunological approach allowed the detection and titration of the glutamate dehydrogenase and aspartate aminotransferase present in high-glutamate affinity proteolipid in inhibited forms, the latter being 26-fold more concentrated than the former.     

The characterization of energized and partially de-energized (respiration-independent) β-galactoside transport into escherichia coli

Unidirectional fluxes of [¹⁴C]lactose by whole cells of Escherichia coli under highly energized and partially de-energized (in the presence of CN^?) conditions are analyzed kinetically.When the cells are energized, the value for $\text{V}$ influx is $\text{0.45 ± 0.01}\text{mM}$ internal concentration increment/s and $\text{K}{}_{\mathrm{t}}$ is $\text{0.26 ± 0.03}\text{mM}$. At an external concentration of 0.61 mM the steady-state internal concentration is 0.25 M, reached after about 1h. The maximum steady-state concentration ratio is $\text{2 · 10}{}^3$.The efflux process under these conditions is non-saturable, being linearly dependent upon internal concentration over the range 25–250 mM with a first-order rate constant of $\text{8.8 ± 0.2 · 10}{}^{\mathrm{?4}}\text{s}{}^{\mathrm{?1}}$.The transport in the presence of CN^? is active, with a maximum concentration ratio (internal concentration/external concentration) of 104, and the uptake is mimicked by anoxia (< 70 ppm O₂).The effects of CN^? are to lower the $\text{V}$ for influx and to change the efflux from a non-saturable to a saturable process with a value for $\text{K}{}_{\mathrm{t}}$ (60 mM) intermediate between that for energized efflux ($\text{> 250}\text{mM}$) and influxe (0.3–0.6 mM), the latter value not changing appreciably. Partial de-energization thus affects both the influx and efflux processes.     

The enzyme "aldehyde oxidase" is an iminium oxidase. Reaction with nicotine delta 1'(5') iminium ion.

The second of the two reaction steps involved in the metabolic transformation of (?)-nicotine to (?)-cotinine $\text{(}\text{3}\text{) (}\text{i.}\text{e.}$, the oxidation of the intermediate $\text{2}\text{)}$ is mediated mainly, if not solely, by the enzyme aldehyde oxidase (EC 1.2.3.1). Of the molecular species that constitute $\text{2}$, nicotine Δ^1′(5′) iminium ion $\text{(}\text{2a}\text{)}$ appears to serve as the substrate. The enzyme has a strong affinity for $\text{2a}$, as shown in a study on the inhibition of the oxidation of 3-(aminocarbonyl)-1-methylpyridinium chloride. This study gave a value of K_i = 6 μM; K_m = 2 μM (pH 7.4). Mainly in view of this finding, “iminium oxidase” seems to be a more adequate name than “aldehyde oxidase” for this enzyme.     

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K⁺ concentration ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{: 200–300}\text{mM}$) which greatly exceeds that of the growth medium, and a low Na⁺ concentration ($\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{: 20}\text{mM}$). Unlike $\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{, K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ varies with cell aging.The K⁺ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K⁺ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ decreases and is partially compensated by a gain in Na⁺. This substitution completely reverses when metabolic substrate is added (K⁺ reaccumulation process). Kinetic analysis of K⁺ movement in cells with steady K⁺ level shows that most of K⁺ influx is mediated by an autologous K⁺-K⁺ exchange mechanism. On the other hand, during K⁺ reaccumulation by K⁺-depleted cells, a different mechanism (a K⁺ uptake mechanism) with higher transport capacity and affinity drives the net K⁺ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K⁺ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent ATPase activity.     

Specificity of d-glucose transport by the apical membrane of Nereis diversicolor epidermis

(1) The specificity of d-[6-³H]glucose influx by a Na⁺-dependent and phlorizin-sensitive transport system in the apical epidermal membrane of the polychaete worm, Nereis diversicolor, was investigated in vivo. (2) The inhibitory effect of eleven d-glucose analogues on d-[6-³H]glucose influx from a 5 μM external concentration was recorded. The inhibitors (each tested at 5, 50, 500 and 5000 μM) were selected to illuminate the configurational requirements for interaction with the d-glucose transport system. (3) The following compounds were found to be significant inhibitors: methyl α-d-glucoside, methyl β-d-glucoside, d-galactose, $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$, 2-deoxy-d-glucose, d-xylose, myo-inositol, β-d-fructose; the effect was graded according to inhibitor concentration. l-Glucose also inhibited d-glucose influx but to the same extent at all four concentrations tested, suggesting transport site heterogeneity. d-Mannose and l-arabinose did not inhibit influx. (4) The most potent inhibitor, methyl-α-d-glucoside, was itself a substrate, and its transport was inhibited by phlorizin and d-glucose, as well as by substitution of Na⁺ in the incubation medium with Li⁺ or choline⁺. (5) We conclude that the specificity of the Na⁺-dependent d-glucose transporter in the apical epidermal membrane of Nereis is similar to that in the apical membrane of vertebrate small intestinal and proximal tubular epithelium, and in the tapeworm integument.     

The effects of vanadate on calcium transport in dialyzed squid axons Sidedness of vanadate-cation interactions

(1) Vanadate (VO₃^?) fully inhibits the ATP-dependent uncoupled Ca efflux (Ca pump) in dialyzed squid axons. (2) Vanadate inhibits with high affinity. The mean apparent affinity ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) obtained was 7 μM. (3) Inhibition by vanadate is dependent on Ca_o. External Ca lead to a release of the inhibitory effect. ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{mM}$). This antagonic effect can be reverted by increasing the vanadate concentration. Internal K⁺ increases the affinity of the intracellular vanadate binding site. External K⁺ has no effect on the inhibition. (4) Vanadate has no effect on the Na_o-dependent Ca efflux component (forward Na-Ca exchange) in the absence of ATP. In axons containing ATP vanadate modified this component.     

Erythrocyte Lii-Nao countertransport system Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Li_i-Na_o countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Li_i-Na_o countertransport. In Na- and Li-free medium, inhibition of Li_i-Na_o countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent K_m  12 mM) is higher than the affinity of Na to its external countertransport site (apparent K_m  25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of $\text{N-}\text{ethyl}\text{[}{}^{14}\text{C}\text{]}\text{maleimide}$ was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.