Facilitated transport of 6-mercaptopurine and 6-thioguanine and non-mediated permeation of 8-azaguanine in novikoff rat hepatoma cells and relationship to intracellular phosphoribosylation

6-Mercaptopurine and 6-thioguanine strongly inhibited the zero-trans entry of hypoxanthine into Novikoff rat hepatoma cells which lacked hypoxanthine/guanine phosphoribosyltransferase, whereas 8-azaguanine had no significant effect. 6-Mercaptopurine was transported by the hypoxanthine carrier with about the same efficiency as its natural substrates (Michaelis-Menten constant = 372 ± 23 μM; maximum velocity = 30 ± 0.7 pmol/μl cell H₂O per s). 8-Azaguanine entry into the cells, on the other hand, showed no sign of saturability and was not significantly affected by substrates of the hypoxanthine/guanine carrier. The rate of entry of 8-azaguanine at 10–100 μM amounted to only about 5% of that of hypoxanthine transport and was related to its lipid solubility in the same manner as observed for various substances whose permeation through the plasma membrane is believed to be non-mediated. Only the non-ionized form of 8-azaguanine (pK_a = 6.6) permeated the cell membrane.Studies with wild type Novikoff cells showed that permeation into the cell was the main rate-determining step in the conversion of extracellular 8-azaguanine to intracellular aza-GTP and its incorporation into nucleic acids. In contrast, 6-mercaptopurine was rapidly transported into cells and phosphoribosylated; the main rate-determining step in its incorporation into nucleic acids was the further conversion of 6-mercaptopurine riboside 5'-monophosphate.     

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.     

Hypoxanthine transport in mammalian cells: Cell type-specific differences in sensitivity to inhibition by dipyridamole and uridine

Summary We have measured by rapid kinetic techniques the zero-trans influx of hypoxanthine in various cell lines and its sensitivity to inhibition by uridine, dipyridamole, nitrobenzylthioinosine and nitrobenzylthiopurine. The results and those reported earlier divided the cells into two distinct groups. In mouse P388, L1210 and L929 cells uridine and hypoxanthine had little effect on the transport of each other, supporting the view that nucleosides and hypoxanthine are transported by different carriers. In these cells, hypoxanthine transport was also uniquely resistant to inhibition by dipyridamole (IC₅₀ (50% inhibition dose) >30M). In Novikoff and HTC rat hepatoma, Chinese hamster ovary and Ehrlich ascites tumor cells, on the other hand, hypoxanthine and uridine inhibited the transport of each other about 50% at a concentration corresponding to the Michaelis-Menten constant of their transport, and hypoxanthine transport was strongly inhibited by dipyridamole (IC₅₀=100 to 400nM). Although these results are compatible with the view that nucleosides and hypoxanthine are transported by a common carrier in these cells, this conclusion is not supported by the finding that uridine transport is strongly inhibited in some of these cell lines, as in first group of cells, by nitrobenzylthioinsine, whereas hypoxanthine transport is highly resistant in all cell lines tested. In contrast, the transport of both substrates is highly resistant to inhibition by nitrobenzylthiopurine. The Michaelis-Menten constants for uridine transport are about the same in all cell lines. The Michaelis-Menten constants for hypoxanthine transport are similar to those for uridine transport in some cell lines, but are much higher in others. This difference is unrelated to the sensitivity of uridine and hypoxanthine transport to inhibition by each other or dipyridamole.     

Adenosine transport and metabolism in mouse leukemia cells and in canine thymocytes and peripheral blood leukocytes.

The intracellular accumulation of free [³H] adenosine was measured by rapid kinetic techniques in P388 murine leukemia cells in which adenosine metabolism (phosphorylation and deamination) was completely prevented by depletion of cellular ATP and by treatment with deoxycoformycin. Nonlinear regression of integrated rate equations on the data demonstrate that the time courses of labeled adenosine accumulation at various extracellular adenosine concentrations in zero-trans and equilibrium exchange protocols are well described by a simple, completely symmetrical, transport model with a carrier:substrate affinity constant of about 150 μM. Adenosine transport was not affected by 1 mM deoxycoformycin indicating that this analog has a low affinity for the nucleoside transport system. The transport capacity of dog thymocytes and peripheral leukocytes was similar to that of P388 cells. Transport was not inhibited by deoxycoformycin and remained constant during the first two hours after mitogenic stimulation with concanavalin A. In untreated, metabolizing P388 cells transport was found to be the major determinant of the rate of intracellular metabolism, regardless of the extracellular adenosine concentration (up to at least 160 μM), but the long-term accumulation (longer than 30–60 seconds) of radioactivity from extracellular adenosine strictly reflected the rate of formation of nucleotides (mainly ATP). The metabolism of adenosine by whole cells was entirely consistent with the kinetic properties of the transport system and those of the metabolic enzymes. At low exogenous adenosine concentrations (1 μM and below) transport was slow enough to allow direct phosphorylation of most of the entering adenosine. The remainder was deaminated and rapidly converted to nucleotides via inosine, hypoxanthine, and IMP. At concentrations of 100 μM or higher, on the other hand, influx exceeded the maximum velocity of adenosine kinase about 100 times so that most of the entering adenosine was deaminated. But since the maximum velocity of adenosine deaminase exceeded those of nucleoside phosphorylase and hypoxanthine/guanine phosphoribosyltransferase about 5 and 100 times, respectively, hypoxanthine and inosine rapidly exited from the cells and accumulated in the medium. A 98% reduction of adenosine transport (at 100 μM), caused by the transport inhibitor Persantin, inhibited adenosine deamination by whole cells to about the same extent as transport, whereas adenosine phosphorylation was relatively little affected; thus in the presence of Persantin, transport and metabolism resembled that occurring at the low adenosine concentration. These and other results indicate that adenosine deamination is an event distinct from transport, which occurs only subsequent to adenosine's transport into the cell.     

Nucleoside transport in cultured mammalian cells multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine

The zero-trans influx of 500 μM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine (NBTI) in a biphasic manner; 60–70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID₅₀ = 3?10 nM) and is designated NBTI-sensitive transport. The residual transport activity, designated NBTI-resistant transport, was inhibited by NBTI only at concentrations above 1 μM (ID₅₀ = 10?50 μM). S49 cells exhibited only NBTI-sensitive uridine transport, whereas Novikoff cells exhibited only NBTI-resistant uridine transport. In all instances NBTI-sensitive transport correlated with the presence of between 7·10⁴ and 7·10⁵ high-affinity NBTI binding sites/cell (K_d = 0.3?1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI-resistant and NBTI-sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI-sensitive and an NBTI-resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI. The latter site seems to be unavailable in NBTI-resistant transporters. The proportion of NBTI-resistant and sensitive uridine transport was constant during proportion of NBTI-resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.     

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.     

Chick kidney microsomal cytochrome P-450 involvement in the metabolism of 25-hydroxyvitamin D3

Suspensions of 2 to 5% rat thymocytes were incubated at 35 °C in buffered balanced salt solution (pH 7.3) with lactate and β-hydroxybutyrate as fuels. The dependence of 3-O-[Me-³H]methylglucose influx on external and internal 3-O-methylglucose concentrations was studied. Entry was almost rectilinear during the first minute. From the dependence of methylglucose entry (into sugar-free cells) on external methylglucose concentration, we judged the entry K_m to be about 7.7 mm and the entry V to be about 0.64 μmol · min^?1 · (ml of packed cell volume)^?1. Methylglucose inside the cell enhanced influx, hence equilibrium exchange was faster than entry. The dependence of equilibrium exchange on methylglucose concentration (inside and outside being equal) indicated a K_m of about 25 mm and a V of about 2.1 μmol · (min)^?1 · (ml of cell volume)^?1. This effect of internal sugar indicated that entry into sugar-free cells is limited mainly by the return of empty carrier to the outside surface and that loading the carrier on the inside enhances its outward mobility. The K_m and V for influx into cells containing 21 mm methylglucose were 5.9 mm and 1.17 μmol · min^?1 · (ml of packed cells)^?1. The effect of 21 mm internal sugar on lowering the influx K_m from about 7.7 mm to about 6 mm was reproducible and contributed to the evaluation of the constants of the transport rate law. It indicated that loading of the carrier at the external surface reduces its mobility, in contrast to the effect of loading on the inside. Mechanical explanations for this behavior are discussed.     

The human erythrocyte ghost: A new experimental model for studying adenosine transport

Previous work on adenosine transport has always had problems with the interference of adenosine metabolism, due to its high metabolic rate and because the enzymes involved are consistently present in most tissues. A new experimental model for studying adenosine transport in human erythrocyte ghosts is presented in this work: Human erythrocyte ghosts were sealed in the presence of erythro-3(2-hydroxynonyl)adenine and P1-P5-di(adenosine)5′-pentaphosphate, inhibitors of adenosine deaminase and adenosine kinase, respectively. These ghosts proved to lack adenosine metabolism when incubated in [U-¹⁴C]adenosine at 10 μm concentration at 37 °C for 60 min. Ghosts were 99.4% sealed in the correct orientation and had constant intracellular water volume. With these characteristics, the erythrocyte ghost preparation has many advantages for studying adenosine transport without adenosine metabolism interference. Adenosine transport was studied following the technique of 24., 25. Experiments to study Zero-trans influx and efflux, equilibrium exchange, and infinite-trans influx and efflux are presented. Adenosine transport did not behave linearly in any of these experimental procedures. Adenosine basic kinetic constants, calculated according to the procedure of Lieb and Stein, were R_1→-2 = 4.1 × 10⁻⁴, R_2→-1 = 3.97 × 10⁻⁴, R_ee = 1.94 × 10⁻⁴, R_oo = 6.08 × 10⁻⁴, K_1→-2 = 125.67 μm, andK_2→-1 = 84.36 μm. Lieb and Stein rejection criteria were used to distinguish a simple pore from a simple carrier. The data accumulated indicate that adenosine transport is carried out by a system that satisfies the criteria used for the simple carrier model. Asymmetric behavior was observed indicating lower affinity of the carrier for adenosine influx, although V_max values for influx and efflux were similar.     

Kinetics of glucose transport in human erythrocytes: zero-trans efflux and infinite-trans efflux at 0°C

The kinetic features of glucose transport in human erythrocytes have been the subject of many studies, but no model is consistent with both the kinetic observations and the characteristics of the purified transporter. In order to reevaluate some of the kinetic features, initial rate measurements were performed at 0°C. The following kinetic parameters were obtained for fresh blood: zero-trans efflux K_m = 3.4 mM, V_max = 5.5 mM/min; infinite-trans efflux K_m = 8.7 mM, V_max = 28 mM/min. For outdated blood, somewhat different parameters were obtained: zero-trans efflux K_m = 2.7 mM, V_max = 2.4 mM/min; infinite-trans efflux K_m = 19 mM, V_max = 23 mM/min. The K_m values for fresh blood differ from the previously reported values of 16 mM and 3.4 mM for zero-trans and infinite-trans efflux, respectively (Baker, G.F. and Naftalin, R.J. (1979) Biochim. Biophys. Acta 550, 474–484). The use of 50 mM galactose rather than 100 mM glucose as the infinite-trans sugar produced no change in the infinite-trans efflux K_m values but somewhat lower V_max values. Simulations indicate that initial rates were closely approximated by the experimental conditions. The observed time courses of efflux are inconsistent with a model involving rate-limiting dissociation of glucose from hemoglobin (Naftalin, R.J., Smith, P.M. and Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235–249). The results presented here support the adequacy of the carrier model to account for the kinetics.     

An analysis of the adequacy of the asymmetric carrier model for sugar transport

In 1972, Lieb, W. R.; Stein, W. D. (Biochim. Biophys. Acta 265, 187–207) in their review of sugar transport in human erythrocytes concluded that the conventional two-state carrier model was inconsistent with the experimental data available at that time. Since then, other papers have appeared which question the validity of the model. In this paper, we give a brief derivation of the equations describing the two-state carrier model, and analyze the predictions of the model in the classical experiments, i.e. zero-trans, infinite-cis, and equilibrium exchange. We show that the estimate of the half saturatiion constant of 2.8 mM for glucose at the inner face of the human red cell membrane for the infinite-cis procedure reported by Hankin, B. L., Lieb, W. R. and Stein, W. D ((1972) Biochim. Biophys. Acta 288, 114–126) is unreliable. We note that all of the other experimental findings are consistent with the asymmetric carrier model.     

Effect of sulfhydryl reagents on nucleoside transport in cultured mammalian cells

Incubation of Novikoff rat hepatoma cells; mouse L929, P388 and L1210 cells; and Chinese hamster ovary cells with sulfhydryl reagents, such as p-hydroxymercuribenzoate or p-hydroxymercuribenzenesulfonate, reduced the zero-trans influx of uridine in a concentration-dependent manner. The sensitivity of uridine transport to inhibition varied somewhat for the cell lines, Chinese hamster ovary cells being the most sensitive. Maximum inhibition by p-hydroxymercuribenzoate occurred in 10–20 min of incubation at 37 °C, and was associated with a decrease in maximum transport velocity without significant change in substrate affinity of the carrier. The development of inhibition of uridine influx correlated with binding of [¹⁴C]p-hydroxymercuribenzoate to the cells. Inhibition of transport also roughly correlated with a decreased binding of 6-nitrobenzylthioinosine to high-affinity binding sites on the cells (presumably representing the nucleoside transporter) without affecting binding affinity. Treatment of cells with p-hydroxymercuribenzenesulfonate reduced uridine influx and efflux to a similar extent. Inhibition of uridine transport and binding of [¹⁴C]p-hydroxymercuribenzoate were readily reversed by incubation of the cells with dithiothreitol. The results indicate that sulfhydryl groups are essential for the functioning of the nucleoside transporter, perhaps for the binding of substrate. Blockage of the sulfhydryl groups results in a reversible inactivation of the carrier. Treatment of the cells with the sulfhydryl reagents also caused a concentration-dependent increase in cell volume, which was readily reversed by incubation of the cells with dithiothreitol but seemed unrelated to the inhibition of nucleoside transport.     

Interaction of glycylglycine and Na+ at the mucosal border of Guinea-pig small intestine: A non-mutual stimulation of transport

Sodium-dependence of glycylglycine (Gly-Gly) influx and stimulation of Na⁺ transport by Gly-Gly were studied in everted sacs, sheet preparations and brush-border membrane vesicles isolated from guinea-pig ileum. Gly-Gly influx was found to be independent of the presence of Na⁺, while Na⁺ transport was stimulated by Gly-Gly as evidenced by increases in transmural potential difference (PD_t), short-circuit current (I_sc) and Na⁺ influx. The change in PD_t (ΔPD_t) induced by Gly-Gly was a saturable function of Gly-Gly concentration, showing a Michaelis-Menten type relationship. The half-saturation concentration for Gly-Gly estimated from the electrical data was nearly identical with that estimated from influx data. At a constant Gly-Gly concentration the relationship between I_sc and Na⁺ concentration was sigmoid, and the Hill coefficient was 1.5. Kinetic analysis according to Garay Garrahan indicates that each Gly-Gly carrier has two equivalent non-interacting binding sites for Na⁺, and that translocation of Na⁺ occurs when the two Na⁺ sites on the carrier loaded with Gly-Gly are occupied by Na⁺. However, our results indicate that the resultant Na⁺ flow is not capable of stimulating Gly-Gly translocation.     

Kinetic relations of the Na-amino acid interaction at the mucosal border of intestine

The relation between unidirectional influxes of Na and amino acids across the mucosal border of rabbit ileum was studied under a variety of conditions. At constant Na concentration in the mucosal bathing solution, amino acid influx followed Michaelis-Menten kinetics permitting determination of maximal influx and the apparent Michaelis constant, K_t. Reduction in Na concentration, using choline as substitute cation, caused an increase in K_t for alanine but had no effect on maximal alanine influx. The reciprocal of K_t was a linear function of Na concentration. Similar results were obtained for valine and leucine and these amino acids competitively inhibited alanine influx both in the presence and in the absence of Na. These results lead to a model for the transport system which involves combination of Na and amino acid with a single carrier or site leading to penetration of both solutes. The model predicts that alanine should cause an increase in Na influx and the ratio of this extra Na flux to alanine flux should vary with Na concentration. The observed relation agreed closely with predicted values for Na concentrations from 5 to 140 mM. These results support the hypothesis that interactions between Na and amino acid transport depend in part on a common entry mechanism at the mucosal border of the intestine.     

The pH dependence of exchange transport of glucose in human erythrocytes

In glucose exchange transport into red blood cells the rate of glucose uptake showed two pH dependent maxima, with the larger at approximately pH 7.5 and the smaller one at pH 3. In the studied pH range the relation between the rate of glucose uptake and the substrate concentration followed Michaelis-Menten kinetics. While the maximal velocity (V) reflected the pH changes of the media, the Michaelis constant (K_m) remained constant. The dissociation constants of the groups of the free carrier and the carrier-glucose complex were the same. The pK of the acidic group was 5.2 and of the basic group 9.5. Glucose was not bound to groups of the carrier which dissociated protons in the pH range of three to nine. By rearranging the equation for the pH dependence of the relative influx a more definitive graphic determination of the pK values was produced.     

Kinetics of nucleoside transport in human erythrocytes. Alterations during blood preservation

The transmembrane equilibration of radiolabeled uridine was measured by rapid kinetic techniques in human erythrocytes from freshly drawn blood and in the same cells during conventional storage of the blood as well as in cells from outdated blood. Our results confirm earlier reports that the maximum velocity of uridine equilibrium exchange (Vee) at 25 degrees C is about 30% lower in outdated than fresh red cells, whereas the opposite is the case for the Michaelis-Menten constant for equilibrium exchange (Kee), and that maximum zero-trans efflux (Vzt21) is about 4-times greater than maximum zero-trans influx (Vzt12) in outdated cells (directional asymmetry), whereas they are about the same in fresh red cells. At 25 degrees C, the nucleoside-loaded carrier of fresh cells moves on the average 6-times more rapidly than the empty carrier, whereas the differential mobility of loaded and empty carrier from outdated cells is about 15-fold. Our results also show that greater efflux than influx in outdated cells is not due to a general leakiness of outdated cells, that the differences in kinetic properties of the transporter developed during the first two weeks of blood storage and that the differences are greatly amplified when transport is measured at 5 degrees C rather than 25 degrees C. At 5 degrees C, the loaded carrier from outdated red cells moves about 325-times more rapidly than the empty carrier and maximum zero-trans efflux exceeds maximum zero-trans influx about 14-times, whereas the transport of fresh cells exhibits directional symmetry just as at 25 degrees C. The changes in kinetic properties of transport induced by temperature and storage are probably related to structural alterations in the plasma membrane and suggest that the operation of carrier is subject to modification by the membrane environment. Other results show that the kinetics of the sugar transport of human red cells is not affected in the same manner by blood storage as those of the nucleoside transporter.     

A Method for Characterizing the Relation between Nutrient Concentration and Flux into Roots of Intact Plants

A method based on the rate of depletion of a nutrient from solution was developed to characterize nutrient flux of plant roots. Nutrient concentration of the solution was measured at a series of time intervals to describe the complete depletion curve. An integrated rate equation, based on a Michaelis-Menten model, was developed and fit to the data of the depletion curve using a least-square procedure. The equation contained values for V_max, the maximum rate of influx; Km, the Michaelis constant; and E, efflux, which were used to describe the relation between solution concentration and net influx rate. Models other than Michaelis-Menten could also be used. The method uses only one plant or group of plants to obtain data over a range of nutrient concentrations, is adapted particularly to the low concentration range, and measures the concentration below which net influx ceases. With this method the plant is in steady state absorption prior to the experiment and continues at this steady state until near the end of the experiment.     

Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of ¹⁴C-labelled efflux of 3-O-methyl-d-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K _1/2=7.9 mM) and its aglucon phloretin (K _1/2=84 μM); with both inhibitors, 3-O-methyl-d-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the d-enantiomers of neutral hexoses, including glucose (K _1/2=48 μM), 3-O-methyl-d-glucose (K _1/2=139 μM), and fructose (K _1/2=730 μM), but not by, for instance, d-allose, and l-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the d-glucose conformation.     

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 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 (Km approximately 5-8 microM) than hypoxanthine (Km approximately 90-120 microM) but maximum fluxes at 25 degrees C were generally 5-10-fold lower for adenine (Vmax approximately 0.6-1.4 pmol/microliters per s) than for hypoxanthine (Vmax approximately 9-11 pmol/microliters 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 or 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.     

Adenosine and tubercidin binding and transport in Chinese hamster ovary and Novikoff rat hepatoma cells

The uptake of adenosine and tubercidin by control and ATP-deleted wild-type and adenosine kinase-deficient cells was measured by rapid kinetic techniques. Adenosine deamination was inhibited by pretreatment with 2-deoxy-coformycin. Control wild-type cells phosphorylated adenosine so rapidly that the kinetics of transport per se could not be assessed unambiguously. ATP depletion and adenosine kinase deficiency did not abolish the conversion of adenosine to nucleotides, but reduced it to such an extent that initial velocities of uptake could be safely construed as transport velocities in both zerotrans and equilibrium exchange modes. The same was true for tubercidin, which was not phosphorylated in adenosine kinase-deficient cells. It accumulated intracellularly, however, to concentrations 50 to 120% higher than those in the extracellular space, apparently due to binding to some intracellular component(s). Binding was not saturated up to a concentration of 200 μM, but seemed to be slow relative to transport. Fits of appropriate integrated rate equations based on the simple carrier model to uptake time courses obtained under these conditions yielded Michaelis-Menten constants for adenosine and tubercidin transport of 100 to 200 μM and maximum velocities of 10 to 30 pmol/μl cell H₂O ? sec, whereas the rate of intracellular phosphorylation was maximal at concentrations between 2 and 8 μM. The first-order rate constant (V_max/K_m) for adenosine phosphorylation, however, seemed to be appreciably higher than that for its transport. This indicates that at physiological concentrations, which fall in the first-order range for both processes, adenosine trapping is very efficient. Adenosine, tubercidin, tricyclic nucleoside, 2′-deoxyadenosine, and 3′-deoxyadenosine all inhibited uridine and thymidine transport to about the same extent, whereas pyrazofurin was signficantly less effective.     

Effects of temperature on the transport of nucleosides in guinea pig erythrocytes

The initial rate of [14C]uridine transport by guinea pig erythrocytes was investigated at different temperatures. At 37, 22, and 10 degrees C the concentration dependence of uridine zero-trans influx and equilibrium exchange influx was resolved into two components; (a) a saturable component which followed simple Michaelis-Menten kinetics and which was inhibited by nitrobenzylthioinosine, and (b) a linear component of low magnitude and insensitive to nitrobenzylthioinosine inhibition. The maximum velocity, Vmax, of zero-trans uridine influx for the saturable transport system was 70-fold higher at 37 than 10 degrees C (1.24, 0.20, and 0.018 mmol/L of cells per hour at 37, 22, and 10 degrees C, respectively). Similarly, the apparent affinity, Km, for zero-trans influx decreased as the temperature was lowered (0.27, 0.066, and 0.038 mM at 37, 22, and 10 degrees C, respectively). In contrast, uridine equilibrium exchange influx was less temperature dependent (Vmax, 2.80, 0.89, and 0.14 mmol/L of cells per hour; apparent Km 0.61, 0.36, and 0.24 mM at 37, 22, and 10 degrees C, respectively). These results demonstrate that the mobility of the empty carrier is impaired to a greater extent than the mobility of the loaded carrier temperature decreased. However, the kinetic constants for zero-trans uridine influx and efflux at 37 degrees C were similar, indicating that the nucleoside transporter exhibited directional symmetry at 37 degrees C. Arrhenius plots of the maximum velocity for equilibrium exchange and zero-trans uridine influx were discontinuous above 25 degrees C, but between 20 and 5 degrees C the plots were linear (Ea = 22 and 30 kcal/mol for equilibrium exchange and zero-trans influx, respectively.