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Biomedical subjects

R M Krupka

Publications and source records attributed to R M Krupka.

At least 37 records · Page 2Linked to original sources
PubMed1988

The choline carrier of erythrocytes: location of the NEM-reactive thiol group in the inner gated channel.

Choline transport across the human erythrocyte membrane is irreversibly inhibited when N-ethylmaleimide (NEM) reacts with a carrier SH group which is located outside the substrate site, and which is exposed in the inward-facing form of the carrier but prevented from reacting in the outward-facing form. The location of the SH group with respect to the membrane has now been determined by studying the dependence of the NEM-alkylation rate on the intracellular and extracellular pH. The results show that the reactive SH group equilibrates with hydrogen ions in the cytoplasm, but is completely isolated from hydrogen ions in the external medium. With this added evidence it becomes possible to conclude that the SH group is located in the inner gated channel of the choline carrier.

Carrier Proteins↗
PubMed1987

Effects on transport of rapidly penetrating, competing substrates: activation and inhibition of the choline carrier in erythrocytes by imidazole.

The properties of the choline transport system are fundamentally altered in saline solution containing 5 mM imidazole buffer instead of 5 mM phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at the cis side inhibits, and at the trans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.

Biological Transport↗
PubMed1986

The carrier reorientation step in erythrocyte choline transport: pH effects and the involvement of a carrier ionizing group.

Under zero-trans conditions, the facilitated transport of choline across the erythrocyte membrane is limited by the rate of reorientation of the free carrier; as a result the pH dependence of this step can be investigated, independent of other steps in transport. It is found that as the pH declines (between 8.0 and 6.0) the rate of inward movement of the free carrier rises and the rate of outward movements falls, so that the partition of the free carrier increasingly favors the inward-facing form. When the pH of the cell interior and of the medium are varied independently, the partition responds to the internal but not the external pH. The membrane potential, which varies somewhat as the pH is altered, has no effect on the carrier partition. The analysis of the results indicates that the carrier mobility is dependent on an ionizing group of pKa 6.8, which is exposed on the cytoplasmic surface of the membrane in the inward-facing carrier; in the outward-facing carrier the ionizing group appears to be masked, in that its pKa is shifted downward by more than one unit. The observations can be explained by assuming that an ionizing group is located in the wall of a gated channel connecting the substrate site with the cytoplasmic face of the cell membrane.

Biological Transport↗
PubMed1986

Looking for probes of gated channels: studies of the inhibition of glucose and choline transport in erythrocytes.

Two seemingly contradictory sets of observations have been made in studies of biological transport, which are essential for our understanding of the transport mechanism: carriers are integral membrane proteins, which span the membrane and are not free to rotate across the membrane; carriers appear to function like a ferryboat, with a substrate binding site moving back and forth from one side of the membrane to the other. To reconcile these facts, it is necessary to postulated gated channels connecting the substrate site with the two membrane surfaces: the channels are arranged so that as one opens the other closes, with the result that the substrate site is alternately accessible from opposite sides of the membrane. Based on these properties, the following distinguishing features of molecules specifically bound in the channels may be predicted: if sufficiently bulky, they inhibit transport; they bind outside the substrate site (though adjacent to it), they bind asymmetrically either to the outward-facing carrier and on the outer surface of the membrane, or to the inward-facing carrier and on the inner surface of the membrane. The asymmetrical inhibition of the glucose and choline transport systems of erythrocytes by various inhibitors is examined, and the behavior in every case is found to conform with these criteria. From the results it may be concluded that the glucose carrier binds cytochalasin B in the inner gated channel and phloretin and tetrathionate in the outer gated channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Asymmetrical binding of phloretin to the glucose transport system of human erythrocytes.

The sidedness of phloretin binding to the glucose carrier has been determined by comparing the type of inhibition produced in zero trans entry and zero trans exit experiments. Initial rates of zero trans entry were measured by the method of R.D. Taverna and R.G. Langdon (Biochim. Biophys. Acta 298:412-421, 1973), which involves pink ghosts loaded with glucose oxidase; this obviates the problem of rapid substrate accumulation inside the cells. With phloretin equilibrated across the membrane, the inhibition of entry was competitive, and the inhibition of exit noncompetitive. The experimental procedures were validated by showing that the inhibition by cytochalasin B, known to bind inside but not outside, was noncompetitive in entry and competitive in exit, as predicted. It was also demonstrated that even after pre-incubation of the cells with a relatively high concentration of phloretin, the phloretin adsorbed in the membrane did not significantly alter the rate of carrier reorientation. The results show that the outward-facing form of the glucose carrier, but not the inward-facing form, bears a phloretin binding site; thus phloretin, as well as cytochalasin B, is bound asymmetrically, phloretin outside and cytochalasin B inside.

Blood Glucose↗
PubMed1985

Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: evidence for inward-facing and outward-facing carrier conformations.

Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1984

The relationship between substrate dissociation constants derived from transport experiments and from equilibrium binding assays. Implications of the conventional carrier model.

A kinetic analysis of substrate and inhibitor binding, based on the conventional carrier model, leads to the following conclusions. The substrate constant derived from equilibrium binding studies is not a simple dissociation constant; rather, it is identical to the half-saturating substrate concentration for equilibrium exchange transport, which is a function of both the dissociation constant and the rate constants for carrier reorientation. In general, binding and transport constants are identical, assuming the same substrate distribution across the membrane in the two experiments. Binding studies reveal only a single substrate site--even if the carrier is unsymmetrical, with different substrate affinities on the two sides of the membrane. The binding constants for inhibitors are identical to the inhibition constants found in transport. These rules, which apply to a carrier imbedded in the cell membrane or free in solution, offer a means of deciding whether an isolated carrier retains the properties of the intact system.

Carrier Proteins↗
PubMed1984

The comparative specificity of the inner and outer substrate transfer sites in the choline carrier of human erythrocytes.

The substrate specificities on the inner and outer surfaces of the cell membrane have been compared by determining the relative affinities, inside and outside, of a series of choline analogs. The results of two different methods were in agreement: (1) the carrier distribution was determined in the presence of a saturating concentration of an equilibrated analog, using N-ethylmaleimide as a probe for the inward-facing carrier; (2) the degree of competition was measured between an equilibrated analog and choline in the external solution. The carrier sites are found to have markedly different specificities: the outer site is more closely complementary to the structure of choline than is the inner, and even a slight enlargement of either the trimethylammonium or hydroxyethyl group gives rise to preferential binding inside. It is also found that a nonpolar binding region, which is adjacent to the outer site, is absent from the inner site. As the transport mechanism involves the exposure of only one site at a time, first on one surface and then the other, it follows that an extensive reorganization of the structure of the substrate site may occur during the carrier-reorientation step, or alternatively that two distinct sites may be present, only one of which is exposed at a time.

Amines↗
PubMed1983

The kinetics of transport inhibition by noncompetitive inhibitors.

A new analysis of the conventional carrier model shows that noncompetitive inhibitors can give rise to either competitive, noncompetitive or uncompetitive kinetics; the true mechanism and also the relative affinity of the inhibitor on each surface of the membrane can be decided from the patterns of inhibition observed in different transport experiments. The principles governing the kinetics of inhibition apply to both reversible and irreversible inhibitors, for in either case the substrate may increase or decrease inhibition or be without effect. Ambiguity arises if the noncompetitive inhibitor acts on only one side of the membrane and if the substrate, in the course of being transported, alters the steady-state distribution of the carrier between inner and outer forms. In facilitated transport systems only equilibrium exchange should give rise to noncompetitive kinetics, whatever the location of the inhibitor. In active systems even the interpretation of exchange in the final steadystate is complicated if the energy-coupling mechanism produces a large displacement in the distribution of the free carrier or the substrate complex: the inhibition could be competitive or uncompetitive, depending on the location of the inhibitor. The actual mechanism is revealed in the uncoupled system.

Biological Transport↗
PubMed1983

Apparent noncompetitive inhibition of choline transport in erythrocytes by inhibitors bound at the substrate site.

According to the conventional carrier model, an inhibitor bound at the substrate transfer site inhibits competitively when on the same side of the membrane as the substrate, but noncompetitively when on the opposite side. This prediction was tested with the nonpenetrating choline analog dimethyl-n-pentyl (2-hydroxyethyl) ammonium ion. In zero trans entry and infinite trans entry experiments, where the labeled substrate and the inhibitor occupy the same compartment, the inhibition was competitive, but in zero trans exit it was noncompetitive, in accord with the model. Similar behavior was seen with dimethyl-n-decyl (2-hydroxyethyl) ammonium ion. With this property of the choline transport system established, it becomes possible to estimate the relative affinity inside and outside of inhibitors present on both sides of the membrane. The tertiary amine, dibutylaminoethanol, which enters the cell by simple diffusion, is such an inhibitor. Here the inhibition kinetics were the reverse of those for nonpenetrating inhibitors; zero trans and infinite trans exit was inhibited competitively, and zero trans entry noncompetitively. It follows that dibutylaminoethanol binds predominantly to the inner carrier form.

Biological Transport↗
PubMed1981

An experimental test for cyclic versus linear transport models. The mechanisms of glucose and choline transport in erythrocytes.

Numerous mechanisms have been suggested to explain transport across biological membranes, all of which fall into one or the other of two distinct categories. In some, substrate sites in the free carrier are simultaneously exposed on the two membrane surfaces, while in others a substrate site is alternately exposed on opposite sides. Either group could account for active and facilitated transport, as well as for accelerated exchange, countertransport, and hyperbolic substrate saturation curves. A simple kinetic test is described here which distinguishes between these two classes. The test depends on measurements of transport rates in the presence of reversible competitive inhibitors inside and outside the cell. Experiments are reported on the glucose system of erythrocytes involving the inhibitors phloretin and cytochalasin B, and on the choline system of the same cells, with the nontransported substrate analogs dimethyl-n-pentyl(2-hydroxyethyl)ammonium ion and 2-di-butylaminoethanol. The results are in agreement with the single site-exposure models, which include the classical carrier, and incompatible with the dual site-exposure models. The mechanisms in the latter group are therefore rejected as explanations for glucose or choline transport.

Biological Transport↗
PubMed1981

Evidence for a two-state mobile carrier mechanism in erythrocyte choline transport: effects of substrate analogs on inactivation of the carrier by N-ethylmaleimide.

Choline transport in erythrocytes is irreversibly inhibited by N-ethylmaleimide. The hypothesis that the carrier alternates between outward-facing and inward-facing forms and that only the latter reacts with the inhibitor (Martin, K. (1971) J. Physiol. (London) 213:647--667; Edwards, P.A. (1973) Biochim. Biophys. Acta 311:123--140) is here subjected to a quantitative test. In this test the effects of a series of substrate analogs upon rates of inactivation and rates of choline exit are compared. By hypothesis the effect of an analog in the external solution on the inactivation rate depends only on how it affects the proportion of the inward-facing carrier. Since 14C-choline efflux is necessarily proportional to the concentration of free carrier in the inward-facing form, the analogs should have related effects on the two rates. In every case the observed effects were identical, whether the analogs accelerated transport or inhibited it. Analysis of the results demonstrates that (1) the transport mechanism depends on the operation of a mobile element; (2) distinguishable inward-facing and outward-facing conformations of the free carrier, carrier-substrate complex, and carrier-inhibitor complex exist, and only the inward-facing forms react at a significant rate with N-ethylmaleimide; (3) carrier mechanisms involving a single form of free carrier or a single form of carrier-substrate complex are ruled out; and (4) dissociation of the carrier-substrate complex is a rapid step with all substrate analogs.

Biological Transport, Active↗
PubMed1981

Reaction of internal forms of the choline carrier of erythrocytes with N-ethylmaleimide: evidence for a carrier conformational change on complex formation.

The choline carrier of human erythrocyte membranes exists in distinguishable outward-facing and inward-facing conformations, and previous studies demonstrated that only the latter reacts with N-ethylmaleimide, producing an irreversible inhibition of transport. We now report experiments to determine the individual reaction rates for the two inward-facing forms: the free carrier and the complex. The pseudo-first-order rate constant for the complex with a substrate analog, di-n-butylaminoethanol, is found to be nearly double that for the free carrier, showing that the carrier conformation is altered following addition of a ligand (with 1 mM N-ethylmaleimide at pH 6.8, 37 degree C, the constants are 0.57 +/- 0.05 min-1 and 0.33 +/- 0.02 min-1, respectively). Hence three different conformational states have been distinguished by experiment: (1) the inward-facing free carrier; (2) the inward-facing complex; and (3) the outward-facing carrier.

Choline↗
PubMed1980

Testing transport systems for competition between pairs of reversible inhibitors. Inhibition of erythrocyte glucose transport by cytochalasin B and steroids.

When two different inhibitors, which are unrelated in structure, inhibit transport by binding to the carrier on the same side of the membrane, it is important for an understanding of the mechanism to decide whether they add to a single common site or to two separate sites. To help resolve this problem, kinetic tests are described for determining whether the inhibitors compete with one another. The test involves measuring the inhibition produced by the inhibitors when acting alone or together. The rate observed with both inhibitors present is then compared with that predicted from the separate inhibitions, for the case of either one or two inhibition sites. The test is applied to the inhibition of glucose transport in erythrocytes by cytochalasin B and the steroids androstenedione and androstanedione. These inhibitors were previously shown to bind to sites which are distinct from the substrate site and which are only present in the inward-facing form of the carrier. Cytochalasin B is now found to compete with the steroids. This finding, together with the earlier evidence that both inhibitors sterically protect the carrier against reaction with fluorodinitrobenzene, leads us to conclude that a single site binds both types of inhibitor.

Androstanes↗
PubMed1980

The electrostatic contribution to binding in the choline transport system of erythrocytes.

Half-saturation constants have been determined for the choline carrier with cationic substrates and their uncharged carbon analogs: (a) choline and 3,3-dimethyl-1-butanol and (b) 2-dimethylaminoethanol and isoamyl alcohol. The constants are 6.3 microM and 16 mM for the first pair, and 19 microM and 45mM for the second. In both cases, the charged molecules have the higher affinity by a factor of more than 2000. This is to be compared with a factor of less than 10 for charged and neutral substrates of acetylcholinesterase, and with a similar factor in antigen-antibody reactions. To account for the unusually strong ionic bond, a very close association between the carrier site and the substrate is suggested, probably with exclusion of water of hydration. This is supported by the fact that gradual replacement of N-methyl groups in the substrate by N-ethyl groups sharply reduces affinity for the carrier with a 110-fold reduction overall, but has no significant effect on the enzymes.

Acetylcholinesterase↗
PubMed1980

The choline transport system of erythrocytes distribution of the free carrier in the membrane.

A method is described, based on the kinetics of transport, for determining the equilibrium distribution of the carrier site on the inner and outer surfaces of the cell membrane, and this method is applied to the choline carrier of human erythrocytes. This method depends on measurement of flux ratios for both entry and exit, i.e., the transport rates of a low concentration of labeled substrate into a solution which contains either no substrate or a saturating concentration of unlabeled substrate. The concentrations of inward-facing and outward-facing carrier are found to be nearly equal, and therefore the 5-fold difference in choline affinity on the inner and outer surfaces of the membrane cannot be explained by an unequal carrier distribution. It is also shown that both reorientation and dissociation of the carrier-substrate complex are far more rapid than reorientation of the free carrier.

Animals↗
PubMed1980

Evidence for allosteric inhibition sites in the glucose carrier of erythrocytes.

2,4-Fluorodinitrobenzene and 2,3-butanedione, which irreversibly inactivate the glucose transfer system of erythrocytes, have been used as probes to determine whether the substrate site and inner and outer sites for reversible inhibitors are located in the same or different regions of the carrier. Inhibitors bound at an inhibition site exposed in the inward-facing but not the outward-facing form of the carrier (cytochalasin B, androstendione and androstandione) protect the transport system against inactivation by 2,4-fluorodinitrobenzene. Inhibitors bound at an external inhibition site (phloretin) and substrates bound at the transfer site do not protect. In contrast inactivation by 2,3-butanedione is slightly accelerated by internally bound inhibitors, while substrates and substrate analogs bound at the transfer site protect the system. It is shown that fluorodinitrobenzene reacts in the inner inhibition site and butanedione in the substrate site; and further that these sites may be separate binding areas in the carrier linked by allosteric interaction. The consequence of this linkage is that binding of a ligand at the substrate site precludes binding of another ligand at the internal or external inhibition site.

Allosteric Site↗