Unloaded Carrier Research Articles

Kinetics of leucine transport in brush border membrane vesicles from lepidopteran larvae midgut.

The kinetics of K(+)-leucine cotransport in the midgut of lepidopteran larvae was investigated using brush border membrane vesicles. Initial rate (3 s) of leucine uptake was determined under experimental conditions similar to those occurring in vivo, i.e. in the presence of delta psi much greater than 0 (inside negative) and a delta pH of 1.4 units (7.4in/8.8out). Leucine and K+ bind to the carrier according to a sequential mechanism, and the binding of one substrate changed the dissociation constant for the other substrate by a factor of 0.15. Both trans-K+ and trans-leucine were mixed-type inhibitors of leucine uptake. Moreover, a portion of total leucine uptake was K+ independent, and it was competitively inhibited by trans-leucine. We interpret the trans inhibitory effects to mean that the partially loaded K+ only form is virtually unable to translocate across the membrane, whereas the binary complex carrier, leucine, can isomerize from the trans to the cis side of the membrane. However, the K(+)-independent leucine uptake occurs with a Keq greater than 1, i.e. the efflux route through the partially loaded leucine only form is slower than the rate of isomerization of the unloaded carrier from trans to cis side. Taken together, these results suggest a model in which transport occurs by an iso-random Bi Bi system. Since K+ does not act as a pure competitive activator, this model is different from that proposed for most of the Na(+)-linked solutes transport agencies and may be related to the broadening of the cation specificity of the amino acid transporters in lepidopteran larvae.

Drug targeting with polyalkylcyanoacrylate nanoparticles:in vitroactivity of primaquine-loaded nanoparticles against intracellularLeishmania donovani

The efficacy of primaquine-loaded polyisohexylcyanoacrylate (PIHCA) nanoparticles was evaluated using J774G8 macrophage-like cells infected with Leishmania donovani: as an in vitro model of visceral leishmaniasis. The in vitro antileishmanial activity of primaquine-loaded nanoparticles showed a 21-fold increase in ED50 compared with free primaquine. Although unloaded PIHCA nanoparticles also exhibited a significant anti-leishmanial effect, the loaded nanoparticles showed a synergistic effect compared with a mixture of unloaded nanoparticles and free primaquine at equivalent concentrations. Primaquine release and isohexanol production were evaluated in a lysosomal fraction; the correlation of both with protein concentration and the rapid drug release indicate the processes are associated with an enzymatic degradation. The results indicate that PIHCA and other polyalkylcyanoacrylates may be useful for targeting drugs at intracellular Leishmania, and that the unloaded carrier itself could be of interest in experimental chemotherapy of leishmaniasis.

Lysine excretion by Corynebacterium glutamicum. 2. Energetics and mechanism of the transport system.

Lysine excretion in Corynebacterium glutamicum was characterized as secondary transport process. It is modulated by three forces: the membrane potential, the chemical potential of lysine, and the proton gradient. The ATP content of the cells did not correlate with the export activity. Lysine is excreted in symport with presumably two OH- ions which is not distinguishable experimentally from an antiport mechanism against two protons. The substrate-loaded carrier is uncharged. When the external substrate concentration is low and no proton gradient present, reorientation of the positively charged, unloaded carrier is rate-limiting. Export then depends on the membrane potential. When the external substrate is high, translocation of the loaded, uncharged carrier is rate-limiting, and export is not modulated by the membrane potential. The lysine secretion system in C. glutamicum is shown to be well adapted to the requirements of metabolite export.

Adenine and hypoxanthine transport in human erythrocytes: distinct substrate effects on carrier mobility

Transport of adenine and hypoxanthine in human erythrocytes proceeds via two mechanisms: (1) a common carrier for both nucleobases and (2) unsaturable permeation 4–5-fold faster for adenine than for hypoxanthine. The latter process was resistant to inactivation by diazotized sulfanilic acid. Carrier mediated transport of both substrates was investigated using zero-trans and equilibrium exchange protocols. Adenine displayed a much higher affinity for the carrier ( K m ≈ 5–8 μM) than hypoxanthine ( K m ≈ 90–120 μM) but maximum fluxes at 25°C were generally 5–10-fold lower for adenine ( V max ≈ 0.6–1.4pmol/μl per s) than for hypoxanthine ( V max ≈ 9–11pmol/μl per s). The carrier behaved symmetrically with respect to influx and efflux for both substrates. Adenine, but not hypoxanthine reduced carrier mobility more than 10-fold. The mobility of the unloaded carrier, calculated from the kinetic data of either hypoxanthine of adenine transport, was the same thus providing further evidence that these substrates share a common transporter and that their membrane transport is adequately described by the alternating conformation model of carrier-mediated transport.

Kinetics of DIDS inhibition of HL-60 cell anion exchange rules out ping-pong model with slippage

According to the ping-pong model of band 3-mediated anion exchange, the transport protein has a single transport site, which can exist in either an inward-facing or an outward-facing conformation. Anions bind to these unloaded forms of the carrier, and translocation takes place only when a suitable anion is bound to the transport site. In a previous paper [Am. J. Physiol. 257 (Cell Physiol. 26): C520-C527, 1989], we had shown that the substrate kinetics of Cl-Cl exchange in the promyelocytic HL-60 cell cannot be explained by this simple ping-pong model of anion exchange but is consistent with a simultaneous model according to which both extracellular and intracellular anions must bind before simultaneous translocation can take place. In the present paper we show that external 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibits anion exchange in HL-60 cells by competing with Cl- for binding to the outward-facing transport site. Furthermore, there is a linear dependence of the slope of the Dixon plot for inhibition by DIDS on the reciprocal of the intracellular Cl- concentration. This result clearly rules out a simple ping-pong scheme. In addition, the data also rule out a ping-pong model in which some translocation of the unloaded carrier is allowed (ping-pong model with slippage). The observed inhibition kinetics can be modeled by a simultaneous model of Cl-Cl exchange with competitive inhibition by DIDS.

Heterogeneity of amino acid transport in horse erythrocytes: a detailed kinetic analysis of inherited transport variation.

1. Thoroughbred horses were divisible into five distinct amino acid transport subgroups on the basis of their erythrocyte permeability to L-alanine, measured uptake rates ranging from 5 to 625 mumol l cells-1 h-1 (0.2 mM-extracellular L-alanine, 37 degrees C). 2. Erythrocytes from animals belonging to the lowest L-alanine permeability subgroup (5-15 mumol l cells-1 h-1) (transport-deficient type) exhibited slow nonsaturable transport of this amino acid. In contrast, cells from horses of the four transport-positive subgroups possessed additional high-affinity (apparent L-alanine Km (Michaelis constant) congruent to 0.3 mM) and/or low-affinity (apparent L-alanine Km congruent to 13 mM) Na+-independent transport routes selective for L-neutral amino acids of intermediate size. The two transporters, designated systems asc1 and asc2, respectively, also possessed a significant affinity for dibasic amino acids. 3. Amino acid transport activity in horse erythrocytes behaved as if controlled by three co-dominant alleles (s, h and l), where s is a silent allele, and h and l code for the functional presence of systems asc1 and asc2, respectively. 4. At physiological temperature, system asc1 operated preferentially in an exchange mode. In contrast, system asc2 did not participate in exchange reactions at 37 degrees C, but did exhibit significant trans-acceleration at 25 degrees C. 5. Reduction of the incubation temperature also resulted in dramatic decreases in apparent Km and Vmax for L-alanine uptake by system asc2, whereas the effects of temperature on system asc1 were much less marked. At 5 degrees C the two transporters exhibited equivalent kinetic constants for L-alanine influx. L-Alanine uptake by transport-deficient cells was relatively insensitive to temperature. Influx by this route may represent the ground-state permeability of the lipid bilayer. 6. The effects of low temperature on system asc2 suggest a preferential impairment of the mobility of the unloaded carrier relative to that of the loaded transporter. Similarly, the different kinetic properties of systems asc1 and asc2 at physiological temperature are attributed to a difference in the mobilities of the empty carriers, this difference being minimized at 5 degrees C.

Kinetics of the intestinal brush border proline (Imino) carrier.

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.

The kinetics of glucose transport in human red blood cells

A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of d-[ 14C]glucose transport into human red blood cells at temperatures ranging from 0° to 53°C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20°C, but Arrhenius plots of maximum d-[ 14C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0°C of 0.726 ± 0.0498 s −1 and 12.1 ± 0.98 s −1 for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 ± 3.47 s −1 and 1113 ± 494 s −1 for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 ± 3.10, 127 ± 4.78, 88.0 ± 6.17 and 31.7 ± 5.11 kJ · mol −1. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward-facing conformations and the affinity for glucose is about 2.5-times greater outside than inside.

48] Fluorescent nucleotide analogs as active site probes for the ADP/ATP carrier and the uncoupling protein

Publisher Summary This chapter focuses on the fluorescent nucleotide analogs as active site probes for the adenosine diphosphate (ADP)/ adenosine triphosphate (ATP) carrier and the uncoupling protein. The 1,5- dimethylaminonaphthoyl (DAN) derivatives DAN-adenine monophosphate (AMP), DAN-ADP, and DAN-ATP have identical fluorescence properties. Their fluorescence is virtually quenched in aqueous solution, whereas a high fluorescence yield is obtained in nonaqueous media, such as dioxane and ethanol. The DAN-nucleotides added to rat liver mitochondria do not give a marked fluorescence. In beef heart mitochondria, they exhibit a relatively small signal which is fully enhanced in submitochondrial (sonic) particles. Also breaking the mitochondria by freezing-thawing or by detergents enhances the fluorescence response. The DAN-nucleotides bind also to the soluble and partially purified ADP/ATP carrier (AAC) with a fluorescence maximum at a slightly longer wavelength, 519 Nm. In the solubilized AAC, the full fluorescence response to addition of DAN-AMP is observed only in the presence of ADP or ATP. This is interpreted to show that the AAC is, from the start, partially in the m-state and partially in the c-state. Only when the substrates for the ADP–ATP transport are provided the transition between the c- and m-state can be accomplished since the unloaded carrier cannot undergo this transition. Thus the carrier population in the c-state is largely converted into the m-state under the pulling force of the DAN-AMP binding.

Concentration dependence of the chloride selfexchange and homoexchange fluxes in human red cell ghosts.

The concentration dependence of the unidirectional chloride flux in human red cell ghosts was studied under selfexchange and under homoexchange conditions. Under selfexchange conditions the intracellular concentration of chloride [Cl]in is equal to the extracellular concentration [Cl]ex and [Cl]in and [Cl]ex are raised concomitantly. Under homoexchange conditions [Cl]in or [Cl]ex were varied separately at a fixed trans-concentration of chloride. The chloride fluxes were calculated from the rate of the tracer efflux and the intracellular chloride. All experiments were executed in isotonic (330 mosM) KCl/K-citrate/sorbitol solutions containing 0-100 mM KCl, 40 mM K-citrate and different concentrations of sorbitol for isoosmotic substitution. The chloride selfexchange and the chloride homoexchange fluxes exhibit a pure saturation kinetics. The halfsaturation constant for the chloride selfexchange was approximately 20 mM, the maximal flux was approx. 3.5 X 10(-4) mol/(min . g cells). The apparent chloride halfsaturation constants from the homoexchange experiments were in the range of 0.9-4.5 mM for the outer and of 5.5-14.5 mM (0 degree C, pH 7.3) for the inner membrane surface, both halfsaturation constants increase with increasing trans-concentrations. At infinite trans-concentrations of chloride, the halfsaturation constant for the outer and the inner membrane surface amounts to approximately 5 mM and approximately 15 mM, respectively. The slope of the double reciprocal plots of flux versus cis-chloride concentration decreases with increasing trans-concentration of chloride. The kinetics of the chloride transport provides evidence for a carrier mediated transport mechanism with a single reciprocating transport site. The translocation of the loaded carrier appears to be much faster than the translocation of the unloaded carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Cation dependence of renal outer cortical brush border membrane L-glutamate transport.

The cation dependence of L-glutamate transport in renal outer cortical brush border membrane vesicles was studied. Both cis sodium and trans potassium were required for optimal L-glutamate flux. Relative to simultaneous sodium (out greater than in) and potassium (in greater than out) gradient conditions, flux was reduced 50-fold by potassium removal and more than 100-fold by sodium removal. The effect of potassium removal in this preparation was markedly larger than that observed in other renal brush border membrane preparations. No other monovalent cation tested was effective in replacing sodium. However, Rb+ and Cs+ and to a lesser degree NH+4 were found to be effective potassium substitutes. Kinetic analysis of the Na/K-dependent component of L-glutamate flux indicated a single transport system of Km = 13 microM and Vmax = 1.3 nmol X min-1 X mg protein-1. Studies of the dependence of L-glutamate flux on potassium and sodium concentrations yielded Hill coefficients of 0.9 and 1.9, respectively, consistent with involvement of one potassium ion and two or more sodium ions in the L-glutamate transport event. Efflux studies indicated that sodium and potassium act at different steps in the transport cycle, sodium to facilitate the translocation of the glutamate-carrier complex, and potassium to facilitate the return of the unloaded carrier. A model for renal Na/K-dependent L-glutamate transport is suggested on the basis of these results.

The lactose/H+ carrier of Escherichia coli: lac YUN mutation decreases the rate of active transport and mimics an energy-uncoupled phenotype.

The Escherichia coli K12 strain X71-54 carries the lac YUN allele, coding for a lactose/H+ carrier defective in the accumulation of a number of galactosides [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414]. Previous studies proposed that the lower accumulation in the mutant be due to a faulty coupling of H+ and galactoside fluxes via the carrier. Immunochemical characterization of the carriers in membranes from mutant and parent strains with an antibody directed against the C-terminal decapeptide of the wild-type carrier leads to the conclusion that the mutant carrier is similar to the wild-type in terms of apparent Mr, C-terminal sequence, and level of incorporation into the membrane. The pH-dependence of galactoside transport was compared in the mutant and the parent. At pH 8.0-9.0, mutant and parent behave similarly with respect to the accumulation of beta-D-galactosyl 1-thio-beta-D-galactoside and to the ability to grow on the carrier substrate melibiose. At pH 6.0, both the maximal velocity for active transport and the level of accumulation of beta-D-galactosyl-1-thio-beta-D-galactoside are lower in the mutant. The mutant also is unable to grow on melibiose at pH 5.5. However, at pH 6.0 and low galactoside concentrations, the symport stoichiometry is 0.90 H+ per galactoside in the mutant as compared with 1.07 in the parent. These observations suggest that symport is normal in the mutant and that the lower rate of transport in the mutant is responsible for the phenotype. At higher galactoside concentrations, accumulation is determined not only thermodynamically but also kinetically, contrary to a simple interpretation of the chemiosmotic theory. Therefore lower rates of active transport can mimic the effect of uncoupling H+ and galactoside symport. Examination of countertransport in poisoned cells at pH 6.0 reveals that the rate constants for the reorientation of the loaded and unloaded carrier are altered in the mutant. The reorientation of the unloaded carrier is slower in the mutant. However, the reorientation of the galactoside-H+-carrier complex is slower for substrates like melibiose, but faster for substrates like lactose. These findings suggest that lactose-like and melibiose-like substrates interact with the carrier in slightly different ways.

Mechanism of proline transport in Escherichia coli K12. II. Effect of alkaline cations on binding of proline to a H+/proline symport carrier in cytoplasmic membrane vesicles.

The substrate binding reaction of the proline carrier was investigated in nonenergized conditions using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The binding activity specifically required both alkaline cations (X+), Na+ and Li+, and protons. The Na+-dependent binding activity was dependent on the proline carrier, which is the product of the putP gene, and was not affected by ionophores and energy transduction inhibitors. The parameters of proline binding were determined by double reciprocal plots in reaction media with various combinations of Na+ and H+ concentrations. The apparent dissociation constant was greatly affected by the Na+ and H+ concentrations of the medium and could be expressed as a combination of the reciprocals of the Na+ and H+ concentrations, while the maximum number of binding sites remained constant. The characteristics of proline binding to the carrier can be explained by a mechanism in which the unloaded carrier forms a carrier/H+/X+ (CH+X+) complex by a random equilibrium and only the CH+X+ complex binds substrate in nonenergized conditions, as proposed for the Na+/H+/glutamate symport carrier of E. coli B ( Fujimura , T., Yamato , I., and Anraku , Y. (1983) Biochemistry 22, 1954-1959).

Driving forces and current-voltage characteristics of amino acid transport in Riccia fluitans

The complete steady-state I–V relationship of α-aminoisobutyric acid transport across the plasmalemma of rhizoid cells from Riccia fluitans has been measured and analysed with special emphasis on α-aminoisobutyric acid equilibrium and saturation conditions. (A) The electrical data show that: (1) the amino acid-induced electrical current saturates after the addition of the amino acid, regardless of the concentration; (2) a steady state is reached 1–2 h after incubation in α-aminoisobutyric acid, but after less that 5 min in the presence of 1 mM CN −; (3) the steady-state I–V characteristic of α-aminoisobutyric acid transport is a sigmoid curve and fairly symmetric in current with respect to the voltage axis; and (4) the equilibrium potential is clearly a function of the amino acid accumulation ratio. It is suggested that the sigmoid curve represents the characteristic of carrier-mediated α-aminoisobutyric acid transport with a voltage-insensitive step, possibly the translocation of the unloaded carrier, rate-limiting. Since under normal conditions the voltage-sensitive rate constant k oi is much greater than k io , it is further suggested that the energy to drive this system is put into the transfer of positive charge from outside to the cytoplasm. (B) Accumulation ratios have been determined by inspection of current-voltage data, and additionally by compartmental analysis on green thalli from Riccia fluitans. Both methods give ratios far too low compared with the thermodynamically possible accumulation of about 10 4. It is suggested that substantial leakages via different non-electrical pathways prevent equilibrium at steady state, and it is concluded that in such leaky systems the thermodynamic equilibrium condition is not suitable for estimating stoichiometries.

GABA efflux from synaptosomes: effects of membrane potential, and external GABA and cations.

Presynaptic GABAergic nerve terminals accumulate gamma-aminobutyric acid (GABA) by a sodium-dependent carrier mechanism in which two Na+ are co-transported with one GABA. We have examined the influence of external GABA and cations on GABA efflux from 3H-GABA loaded rat brain synaptosomes, to determine whether or not the carriers can also mediate GABA efflux. We observed that, in Ca-free media (to minimize Ca-dependent, evoked release), external GABA promotes GABA efflux when the medium contains Na+, but inhibits GABA efflux in the absence of Na+. The efflux of GABA into Ca-free media is stimulated by depolarization (either with veratridine or increased external K+). These data, and published data on the internal Na+ dependence of GABA efflux into Ca-free media, indicate that exiting GABA is cotransported with Na+. The stimulatory effect of depolarization is consistent with efflux of Na+ along with the uncharged GABA. The (carrier-mediated) efflux is also stimulated when the carriers cycle inward with Na+ + GABA. The inhibitory effect of GABA in Na+-free media implies that GABA can bind to unloaded carriers and that the carriers loaded only with GABA cycle very slowly, if at all. Our data, and data from the literature, can be fitted to a simple model with sequential binding of solutes: external GABA binds to the carrier first, and only the free or fully-loaded (with 2Na+ + 1GABA) carriers can cycle. Other binding sequences and random binding, do not fit the experimental observations.

The transport of chloroquine across human erythrocyte membranes is mediated by a simple symmetric carrier

The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25°C. Under zero- trans conditions the system displays complete symmetry, the Michaelis constants being 39.2±2.4 μM for influx and 36.6±5.6 μM for efflux. The respective maximal velocities are 206.4±36.0 μM·min −1 and 190.0±7.8 μM·min −1. Under equilibrium-exchange conditions the Michaelis constant is 108.6±15.6 μM and the maximal velocity is 630.3±50.4 μM·min −1. This 3-fold increase in both K and V over the zero- trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.

Na +/solute symport in membrane vesicles from Bacillus alcalophilus

The characteristics of α-aminoisobutyric acid translocation were examined in membrane vesicles from obligately alkalophilic Bacillus alcalophilus and its non-alkalophilic mutant derivative, KM23. Vesicles from both strains exhibited α-aminoisobutyric acid uptake upon energization with ascorbate and N, N, N′, N′-tetramethyl- p-phenylenediamine. The presence of Na + caused a pronounced reduction in the Km for α-aminoisobutyric acid in wild-type but not KM23 vesicles; the maximum velocity ( V) was unaffected in vesicles from both strains. Passive efflux and exchange of α-aminoisobutyric acid from wild-type vesicles were Na +-dependent and occurred at comparable rates (with efflux slightly faster than exchange). This latter observation suggests that the return of the unloaded carrier to the inner surface is not rate-limiting for efflux. The rates of α-aminoisobutyric acid efflux and exchange were also comparable in KM23 vesicles, but were Na +-independent. Furthermore, in vesicles from the two strains, both efflux and exchange were inhibited by generation of a transmembrane electrochemical gradient of protons, outside positive. This suggests that the ternary complex between solute, carrier, and coupling ion bears a positive charge in both strains even though the coupling ion is changed. Evidence from experiments with an alkalophilic strain that was deficient in l-methionine transport indicated that the porters, i.e., the solute-translocating elements, used by non-alkalophilic mutants are not genetically distinct from those used by the alkalophilic parent; that is, the change in coupling ion cannot be explained by the expression of a completely new set of Na +-independent, H +-coupled porters upon mutation of B. alcalophilus to non-alkalophily.

Proton-sulfate co-transport: mechanism of H+ and sulfate addition to the chloride transporter of human red blood cells.

Proton and sulfate inhibition of the obligatory chloride-chloride exchange of human erythrocytes was measured at 0 degrees C to determine their mechanism of reaction with the anion transporter. The proton and sulfate that are co-transported by this mechanism at higher temperatures behaved as nontransported inhibitors at 0 degrees C. We analyzed the data in terms of four molecular mechanisms: (1) HSO4- addition to the transporter; (2) ordered addition with the proton first; (3) ordered addition with the sulfate first; (4) random addition to the transporter. The Dixon plots of 1/MCl vs. [SO4] at different proton concentrations were not parallel. Thus protons and sulfate ions were not mutually exclusive inhibitors. The slope of these Dixon plots was independent of pH above 7.0, which indicates that sulfate could bind to the unprotonated carrier and excludes the first two mechanisms. Protons were inhibitors of chloride flux in the absence of sulfate, which indicates that protons could bind to the unloaded carrier and excludes mechanism 3. The KI for sulfate was 4.35 +/0 0.36 mM. The pK for the protonatable group was 5.03 +/- 0.02. The binding of either a proton or sulfate to the carrier decreased the KI of the other by ninefold. The only simple mechanism consistent with the data is a random-ordered mechanism with more transporters loaded with a sulfate than loaded with a proton at the pH and sulfate concentrations of plasma.

Trans effects in cell membrane transport

The efflux of a substrate from preloaded cells may be decelerated by an inhibitor in the external medium or accelerated by a compatible substrate in the external medium. The derivations of rate equations for the initial velocity of the zero- trans reaction, trans efflux inhibition, and accelerated exchange diffusion are described for steady state conditions. The rate constants making up the Michaelis constant for the trans inhibition reaction are the same as the corresponding parameters in the zero- trans reaction. The rate constants making up the Michaelis constant for the accelerated exchange reaction, however, are different from the corresponding parameters in the zero- trans reaction. The rate equation for trans inhibition shows that the velocity constant for recovery of the unloaded carrier may be determined with steady state experimental data. It is suggested that the observed recovery constant is independent of the substrates and trans inhibitors chosen for an assay of a particular carrier system. An experiment is briefly described to show a determination of a tentative value for the recovery constant of the unloaded nucleoside carrier in yeast cells and the apparent inhibition constant for a trans inhibitor.

Glucose transport characteristics of quiescent thymocytes.

Rat thymocyte populations contain "active" cells which equilibrate quickly with 3-O-methylglucose and "quiescent" cells which equilibrate slowly. Glucose transport stimuli make quiescent cells behave like active cells as regards glucose transport. Glucose transport in quiescent cells was inhibited by phloretin and cytochalasin B. Active cells were heterogeneous in their susceptibility to inhibition by 0.1 micron cytochalasin B, half of them being inhibited about 95%, while half were inhibited modestly. The methylglucose entry Km of quiescent cells was 3 times that of active and stimulated cells. The methylglucose entry Vmax of quiescent cells was one-eighth that of stimulated cells. A fixed internal methylglucose concentration increased the influx Km of quiescent cells but not that of active or stimulated cells. These findings and others are compatible with a model in which the carrier of quiescent cells is masked by an alteration (such as ligand binding) which is relatively specific for empty as opposed to sugar-loaded carrier and rather indifferent as to in versus out carrier orientation. The masking alteration stabilizes inward-facing and outward-facing unloaded carrier against both translocation and loading. The data fail to show evidence of a masking alteration of loaded carrier.