Equilibrium Exchange Experiments Research Articles

Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.

PH and urea dependence of amide hydrogen-deuterium exchange rates in the beta-trefoil protein hisactophilin.

Amide hydrogen/deuterium exchange rates were measured as a function of pH and urea for 37 slowly exchanging amides in the beta-trefoil protein hisactophilin. The rank order of exchange rates is generally maintained under different solution conditions, and trends in the pH and urea dependence of exchange rates are correlated with the rank order of exchange rates. The observed trends are consistent with the expected behavior for exchange of different amides via global and/or local unfolding. Analysis of the pH dependence of exchange in terms of rate constants for structural opening and closing reveals a wide range of rates in different parts of the hisactophilin structure. The slowest exchanging amides have the slowest opening and closing rates. Many of the slowest exchanging amides are located in trefoil 2, but there are also some slow exchanging amides in trefoils 1 and 3. Slow exchangers tend to be near the interface between the beta-barrel and the beta-hairpin triplet portions of this single-domain structure. The pattern of exchange behaviour in hisactophilin is similar to that observed previously in interleukin-1 beta, indicating that exchange properties may be conserved among beta-trefoil proteins. Comparisons of opening and closing rates in hisactophilin with rates obtained for other proteins reveal clear trends for opening rates; however, trends in closing rates are less apparent, perhaps due to inaccuracies in the values used for intrinsic exchange rates in the data fitting. On the basis of the pH and urea dependence of exchange rates and optical measurements of stability and folding, EX2 is the main exchange mechanism in hisactophilin, but there is also evidence for varying levels of EX1 exchange at low and high pH and high urea concentrations. Equilibrium intermediates in which subglobal portions of structure are cooperatively disrupted are not apparent from analysis of the urea dependence of exchange rates. There is, however, a strong correlation between the Gibbs free energy of opening and the denaturant dependence of opening for all amides, which suggests exchange from a continuum of states with different levels of structure. Intermediates are not very prominent either in equilibrium exchange experiments or in quenched-flow kinetic studies; hence, hisactophilin may not form partially folded states as readily as IL-1 beta and other beta-trefoil proteins.

Transport of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) across the human intestinal Caco-2 cell monolayer: role of efflux pumps.

Cooked-food mutagens formed when frying meat have been suggested to contribute to the etiology of colon, breast and prostate cancer. The most prevalent of these mutagens is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which after absorption is bioactivated by both phase I and phase II enzymes. Although available data suggest absorption of PhIP in humans, the extent and mechanism of absorption are unknown. In the present study we examined the transport of [(3)H]PhIP through the human Caco-2 intestinal epithelial cell monolayer, a well-accepted model of human intestinal absorption. The influx, or absorption, was extensive and linear for 2 h and up to a PhIP concentration of 5 microM. Still, the basolateral to apical efflux [apparent permeability coefficient (P(app)) 54.2 +/- 0.7x10(-6) cm/s, mean +/- SEM, n = 24] was 3.6 times greater than the apical to basolateral influx (P(app) 15.1 +/- 0.6x10(-6) cm/s, n = 21, P < 0.0001). Equilibrium exchange experiments demonstrated the efflux to be a true active process. Preincubations with verapamil, an inhibitor of P-glycoprotein-mediated transport, or MK-571, an inhibitor of multidrug resistance-associated protein-mediated transport, stimulated influx and reduced efflux of PhIP, suggesting that PhIP is a substrate for both of these transporters. These findings should be considered when determining exposure to the cooked food mutagens.

Modeling facilitative sugar transporters: transitions between single and double ligand occupancy of multiconformational channel models explain anomalous kinetics.

The four-state simple carrier model (SCM) is employed to describe ligand translocation by diverse passive membrane transporters. However, its application to systems like facilitative sugar transporters (GLUTs) is controversial: unidirectional fluxes under zero-trans and equilibrium-exchange experimental conditions fit a SCM, but flux data from infinite-cis and infinite-trans experiments appear not to fit the same SCM. More complex kinetic models have been proposed to explain this "anomalous" behavior of GLUTs, but none of them accounts for all the experimental findings. We propose an alternative model in which GLUTs are channels subject to conformational transitions, and further assume that the results from zero-trans and equilibrium-exchange experiments as well as trans-effects corresponds to a single-occupancy channel regime, whereas the results from the infinite-cis and infinite-trans experiments correspond to a regime including higher channel occupancies. We test the plausibility of this hypothesis by studying a kinetic model of a two-site channel with two conformational states. In each state, the channel can bind the ligand from only one of the compartments. Under single-occupancy, for conditions corresponding to zero-trans and equilibrium-exchange experiments, the model behaves as a SCM capable of exhibiting trans-stimulations. For a regime including higher degrees of occupancy and infinite-cis and infinite-trans conditions, the same channel model can exhibit a behavior qualitatively similar to a SCM, albeit with kinetic parameters different from those for the single-occupancy regime. Numerical results obtained with our model are consistent with available experimental data on facilitative glucose transport across erythrocyte membranes. Hence, if GLUTs are multiconformational channels, their particular kinetic properties can result from transitions between single and double channel occupancies.

Non-sialate inhibitor of influenza A/WSN/33 neuraminidase.

An N1 strain of influenza A virus neuraminidase (A/WSN/33 NA) was purified and used to screen for inhibitors. As a result, a well-known tuberculostatic, 4'-formylacetanilide thiosemicarbazone (or thiacetazone), was identified. Thiacetazone is a non-sialate compound and inhibits the enzyme in a noncompetitive manner with respect to the substrate sialic acid. Mechanistic studies indicate that the inhibition was due to the competition of thiacetazone with Ca2+, which maintains N1 neuraminidase in an active conformation. The Ki for the inhibition was estimated to be about 4 microM. Equilibrium exchange experiments revealed that when purified A/WSN/33 NA was incubated with 5 microM 45CaCl2, 2 mol of 45Ca2+ ion was exchanged into each mole of NA tetramer and subsequently displaced from the enzyme upon the introduction of the inhibitor. Inhibition of plaque formation by thiacetazone in an MDCK cell culture that had been infected with the influenza A/WSN/33 virus was demonstrated. Thiacetazone was highly specific for A/WSN/33 neuraminidase, since little effect was noted when it was tested against NAs from the other strains of influenza virus or from bacteria. This compound might represent a group of non-sialate inhibitors of influenza NA that bind to a noncatalytic or an allosteric site on the enzyme.

Kinetics of lactate transport in sarcolemmal giant vesicles obtained from human skeletal muscle.

We developed a method that allows the measurement of muscle lactate transport in humans. The transport studies were carried out with giant (1.8- to 36-microns-diam) sarcolemmal vesicles obtained by collagenase treatment of needle biopsy material. Marker enzyme analyses demonstrated that the vesicular membrane is predominantly of sarcolemmal origin, contamination with sarcoplasmic reticulum membranes is very low, and mitochondrial membranes are not a major contaminant. The vesicles were loaded with labeled lactate, and the efflux was measured. The system displayed saturation kinetics and inhibitor sensitivity. In equilibrium exchange experiments (pH 7.4, 21 degrees C), the Michaelis-Menten constant (Km) for the carrier-mediated flux was 30 +/- 8 (SD) mM and maximal transport rate (Vmax) was 184 +/- 24 pmol.cm-2.s-1 (142 nmol.mg protein-1.min-1). In zero-trans efflux experiments, Km was 24 +/- 8 mM and Vmax was 81 +/- 11 pmol.cm-2.s-1 (63 nmol.mg protein-1.min-1). In infinite-cis experiments with a variable lactate concentration on the outside of the vesicles, Km was 8 +/- 4 mM and Vmax was 136 +/- 9 pmol.cm-2.s-1 (105 nmol.mg protein-1.min-1). Thus, the system displayed transacceleration. Low pH (6.4) had no significant effect on equilibrium exchange experiments, whereas in zero-trans experiments low pH at the trans side inhibited the flux by 50%. We concluded that lactate transport can be studied in giant vesicles obtained from a single human muscle biopsy. Our data provide evidence for the existence of a lactate carrier in human sarcolemma. This transport system must be taken into account in models of human lactate kinetics.

Muscle lactate transport studied in sarcolemmal giant vesicles: dependence on fibre type and age

Lactate transport was studied in sarcolemmal giant vesicles obtained from rat or rabbit skeletal muscle. With this technique it is possible to obtain quantitative information on sarcolemmal transport characteristics. In equilibrium exchange experiments with 10, 30 and 60 mM lactate, vesicles from 'red' rat muscles had a 50% higher lactate transport capacity than vesicles from 'white' muscles. Giant vesicles made from rabbit red muscles had a 39% higher lactate transport capacity than vesicles from white muscles. These differences probably reflect a different number of lactate transporters, whereas the lactate affinity in red and white muscles are identical. Lactate transport capacity decreased with age. Sarcolemmal giant vesicles made from 22-month-old rats had a 28% lower transport capacity than vesicles from 2-month old rats. In absolute terms, the initial exchange flux with 30 mM lactate was 92 and 127 pmol cm-2 sec-1 for old and young rats, respectively. In supplementary studies in which microelectrode measurements were made in single mouse muscle fibers, it was shown that the cellular acidification rate due to lactate incubation, was 38% lower in fibres from 18-month old mice than in fibres from 2-month old mice.

Muscle lactate transport studied in sarcolemmal giant vesicles

Lactate transport was studied in giant (median diameter 6.3 μm) sarcolemmal vesicles obtained by collagenase treatment of rat skeletal muscle. The lactate transport displayed stereospecificity, had a high temperature coefficient, and could be inhibited up to 90% with known transport inhibitors (PCMBS and cinnamate). In equilibrium exchange experiments, the l-lactate flux demonstrated saturation kinetics with K m = 23.7 mM and V max = 108pmol cm −2 s −1. With lactate present on only one side of the membrane, (zero trans conditions), V max was reduced to 48 pmol cm −2 s −1. The flux rate displayed transacceleration. The lactate flux was coupled to a parallel H + flux. Under equilibrium exchange conditions, the carrier-mediated lactate flux was not pH-dependent. In the zero trans experiments, H + on the trans side acted as an inhibitor. The loaded form of the carrier reorients faster than the unloaded form, and the protonated form with no lactate bound reorients slowly or is immobile. When compared to intact muscles, the giant sarcolemmal vesicles retain their transport characteristics both qualitatively and quantitatively.

The vasopressin-regulated urea transporter in renal inner medullary collecting duct

The terminal part of the inner medullary collecting duct (terminal IMCD) is unique among collecting duct segments in part because its permeability to urea is regulated by vasopressin. The urea permeability can rise to extremely high levels (greater than 100 x 10(-5) cm/s) in response to vasopressin. Recent studies in isolated perfused IMCD segments have established that the rapid movement of urea across the tubule epithelium occurs via a specialized urea transporter, presumably an intrinsic membrane protein, present in both the apical and basolateral membranes. This urea transporter has properties similar to those of the urea transporters in mammalian erythrocytes and in toad urinary bladder, namely, inhibition by phloretin, inhibition by urea analogues, saturation kinetics in equilibrium-exchange experiments, and regulation by vasopressin. The urea transport pathway is distinct from and independent of the vasopressin-regulated water channel. The increase in transepithelial urea transport in response to vasopressin is mediated by adenosine 3',5'-cyclic monophosphate and is associated with an increase in the urea permeability of the apical membrane. However, little is known about the physical events associated with the activation or insertion of urea transporters in the apical membrane. Because of the importance of this transporter to the urinary concentrating mechanism, efforts toward understanding its molecular structure and the molecular basis of its regulation appear to be justified.

Testing transport models and transport data by means of kinetic rejection criteria.

In the case of a transport system obeying Michaelis-Menten kinetics, completely general relationships are shown to exist between the final ratio of internal and external substrate concentrations, alpha, and the V/Km ratios found in zero-trans-entry, zero-trans-exit and equilibrium-exchange experiments (where V is a maximum substrate flux and Km a substrate half-saturation constant). The proof depends on a new method of derivation proceeding from the form of the experimental data rather than, as has been the practice in kinetic analysis, from a hypothetical reaction scheme. These general relationships, which will be true of all mechanisms giving rise to a particular type of behaviour (here Michaelis-Menten kinetics), provide a test for internal consistency in a set of experimental data. Other relationships, which are specific, can be derived from individual reaction schemes, with the use of traditional procedures in kinetic analysis. The specific relationships include constants for infinite trans entry and exit in addition to constants involved in the general relationships. In conjunction, the general and specific relationships provide a stringent test of mechanism. A set of results that fails to satisfy the general relationships must be rejected; here systematic error or unexpected changes in the transport system in different experiments may have distorted the calculated constants, or the system may not actually obey Michaelis-Menten kinetics. Results in accord with the general relationships, on the other hand, can be applied in specific tests of mechanism. The usefulness of the theorem is illustrated in the cases of the glucose-transport and choline-transport systems of erythrocytes. Experimental results taken from several studies in the literature, which were in accord with hyperbolic substrate kinetics, had previously been shown to disagree with relationships derived for the carrier model, and the model was rejected. The new analysis shows that the data violated the general relationships and therefore cannot decide the issue. More recent results on the glucose-transport system satisfy the general relations and agree with the carrier model.

Reassessment of insulin effects on the Vmax and Km values of hexose transport in isolated rat epididymal adipocytes.

Effects of insulin on the kinetic parameters of hexose transport in rat epididymal adipocytes were re-examined. The transport activity was assessed by measuring the rate of uptake of 3-O-[3H]methyl-D-glucose (MeGlc) under equilibrium exchange and zero-trans conditions. The incubation was carried out at 37 degrees C in an infant incubator. During the incubation, the cell suspension (25%, v/v, in a total volume of 48 microliter) was mechanically swirled at a rate of 600 rpm (r = 2 mm). The swirling facilitated the rapid uptake of MeGlc without stimulating the basal transport activity by "mechanical agitation". The basal and insulin-treated cells were incubated under identical conditions, except for the length of the incubation period. The incubation was terminated by the addition of 350 microliters of 1 mM phloretin, which inhibited transport in approximately 0.06 s. The time course of MeGlc uptake was consistent with the view that the process was a multiple-phase reaction. The initial phase of the reaction was completed when the intracellular distribution space of MeGlc was approximately 1% of the total cell volume. Insulin (10 nM) increased the Vmax value of MeGlc uptake 16-fold in equilibrium exchange experiments and 18-fold in zero-trans experiments. At the same time, the hormone decreased the Km value of MeGlc uptake from 11.7 to 5.4 mM in equilibrium exchange experiments and from 9.7 to 4.8 mM in zero-trans experiments. It is concluded that the major effect of insulin on MeGlc uptake is to increase the Vmax value, but the hormone has the additional effect of lowering the apparent Km value.

Two-carrier models for mediated transport. II. Glucose and galactose equilibrium exchange experiments in human erythrocytes as a test for several two-carrier models

1. 1. Equilibrium exchange of glucose and of galactose is measured in human erythrocytes over wide concentration ranges. One-site Michaelis-Menten-type kinetics are shown for the two sugars. 2. 2. In view of the results, two of the two-carrier models discussed in paper I (Eilam, Y. (1975) Biochim. Biophys. Acta 401, 349–363), the different sequential carriers and the antiparallel simultaneous carriers, are rejected for the sugar transfer system. The antiparallel sequential carriers model is consistent with these results.