Biography:

In the past Robert M. Wohlhueter has collaborated on articles with Jon C. Graff and Peter G.W. Plagemann. One of their most recent publications is Thymidine transport in cultured mammalian cells. Kinetic analysis, temperature dependence and specificity of the transport system. Which was published in journal Biochimica et Biophysica Acta (BBA) - Biomembranes.

More information about Robert M. Wohlhueter research including statistics on their citations can be found on their Copernicus Academic profile page.

Robert M. Wohlhueter's Articles: (20)

Thymidine transport in cultured mammalian cells. Kinetic analysis, temperature dependence and specificity of the transport system

AbstractThe transport of thymidine has been characterized kinetically and thermodynamically in Novikoff rat hepatoma cells grown in culture and, less extensively, in mouse L cells, Chinese hamster ovary cells, P388 murine leukemia cells and HeLa cells. That the characterizations pertained to the transport system per se was ensured, (i) by employing recently developed methods for rapid sampling of cell/substrate mixtures in order to follow isotope movements within a few seconds after initial exposure of cells to substrate; (ii) by utilizing cells rendered, by genetic or chemical means, incapable of metabolizing thymidine; and, (iii) by demonstrating conformity of the transport data to an integrated rate equation derived for a simple, carrier-mediated system. The results indicate that thymidine is transported into mammalian cells by a functionally symmetrical, non-concentrative system for which the carrier : substrate dissociation constant ranges from about 100 μM in Chinese hamster ovary cells, to 230 μM in Novikoff hepatoma cells. In all cell lines investigated, the velocity of transport was sufficient to nearly completely equilibrate low concentrations of thymidine across the membrane within 15 s. Temperature dependence of transport velocity and substrate : carrier dissociation were continuous (EA = 18.3 kcal/mol, ΔH0′ = 9.3 kcal/mol, respectively), and showed no evidence of abrupt transitions. Several natural and artificial nucleosides and nucleic acid based inhibited influx of radiolabeled thymidine, apparently by competing with thymidine for the transport carrier.

Hexose transport in novikoff rat hepatoma cells a simple carrier with directional symmetry, but variable relative mobilities of loaded and empty carrier

AbstractThe kinetics of transport of the non-metabolizable hexose, 3-O-methyl-d-glucose, have been measured in Novikoff rat hepatoma cells by both zero-trans entry and equilibrium exchange procedures. Transport conformed to a simple carrier model which operates symmetrically with respect to direction, but with greater mobility of the loaded than of the empty carrier. Although a complete kinetic description of the transporter can, in theory, be obtained by application of integrated equations describing the time course of substrate equilibrium across the membrane beginning from the zero-trans situation, statistical analysis of hypothetical data indicated that directional asymmetry or differential mobilities of loaded and empty carrier cannot be discerned reliably from such data alone. The difference in mobility of loaded and empty carrier, apparent in a comparison of zero-trans entry and exchange data, ranged from 1.5–7-fold in different batches of cells. It is concluded that the magnitude of the difference is not an inherent property of the transporter, but is determined physiologically, and may be involved in regulation of hexose transport.

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

AbstractThe 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 (Kd = 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.

Regular paperPurine and pyrimidine transport and permeation in human erythrocytes

AbstractTime courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 μM in suspensions of human erythrocytes at 25 or 30°C. At concentrations above 25 μM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the ifnlux and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 μM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations 1 μM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.

Mobility of nucleoside transporter of human erythrocytes differs greatly when loaded with different nucleosides

AbstractTime courses of transmembrane equilibration of 2-chloroadenosine, 2′-deoxyadenosine, 3′-deoxyadenosine, cytidine and 2′-deoxycytidine were measured by rapid kinetic techniques in human erythrocytes under equilibrium exchange and zero-trans conditions. The kinetic parameters for transport were computed by fitting appropriate integrated rate equations to the data pooled for seven concentrations and compared to the kinetic parameters for uridine, adenosine, thymidine and formycin B transport determined previously for human erythrocytes under comparable experimental conditions. The transport of all nucleosides conformed to the simple carrier model and was directionally symmetric. The Michaelis-Menten constants for equilibrium exchange (Kee) ranged from 22 μM for 2-chloroadenosine to about 4 mM for cytidine and the maximum velocities (Vee) differed in a similar manner, so that the first-order rate constants (Vee/Kee) were similar for all nucleosides. The kinetic parameters for 2′-deoxyadenosine transport were similar to those for adenosine transport, whereas the lack of the 3′-OH group greatly reduced the affinity of 3t́-deoxyadenosine (cordycepin) for the carrier, 2′,3′-Dideoxynucleosides were transported < 1% as efficiently as 2′- and 3t́-deoxynucleosides. Thus, the 2′- and 3′-OH groups play an important role in nucleoside transport. The mobility of the carrier when loaded with pyrimidine nucleosides (reflected by Vee was 5–10-times greater than that of the empty carrier, whereas the mobility of th adenosine-loaded or 2′-deoxyadenosine-loaded carrier was about equal to that of the empty carrier. Loading the carrier with 2-chloroadenosine or 3′-deoxyadenosine actually decreased its mobility. Thus, the differential mobility of the loaded and empty carrier differs greatly with the nucleoside substrate. The mobility of the loaded carrier as well as Kee increased with a decrease in lipid solubility of the nucleoside substrate, but the relationship was complex.

Research paper2-Deoxycoformycin inhibition of intracellular phosphorylation of adenosine in Novikoff rat hepatoma cells

AbstractTreatment of cultured wild type and azaguanine-resistant Novikoff rat hepatoma cells with 0.1 to 100 μM 2-deoxycoformycin resulted in an inhibition of more than 50 per cent of the incorporation of 100 μM [8-3H]adenosine into intracellular ATP and nucleic acids. In wild type cells, part of the effect resulted from an inhibition of adenosine deamination of deoxycoformycin, with a consequent decrease of incorporation of radioactivity from adenosine via inosine → hypoxanthine → IMP. This pathway, however, was blocked in azaguanine-resistant cells because of the lack of hypoxanthine/guanine phosphoribosyltransferase. The inhibition of adenosine incorporation by deoxycoformycin in these cells was not mediated at the level of adenosine transport or phosphorylation of AMP. We conclude, therefore, that the intracellular phosphorylation of adenosine is impaired in deoxycoformycin-treated cells. There was a close correlation between inhibition of adenosine deamination and adenosine incorporation, both with respect to effective concentrations of deoxycoformycin and to irreversibility of the inhibition. In addition, intracellular concentrations of adenosine above 1–5 μM were found to strongly inhibit the phosphorylation of adenosine in sity, reflecting substrate inhibition of adenosine kinase. The results indicate that the inhibition of adenosine phosphorylation in deoxycoformycin-treated cells was caused by the accumulation of free adenosine in these cells due to adenosine deaminase inhibition.

Thymidine transport in cultured mammalian cells. Kinetic analysis, temperature dependence and specificity of the transport system

AbstractThe transport of thymidine has been characterized kinetically and thermodynamically in Novikoff rat hepatoma cells grown in culture and, less extensively, in mouse L cells, Chinese hamster ovary cells, P388 murine leukemia cells and HeLa cells. That the characterizations pertained to the transport system per se was ensured, (i) by employing recently developed methods for rapid sampling of cell/substrate mixtures in order to follow isotope movements within a few seconds after initial exposure of cells to substrate; (ii) by utilizing cells rendered, by genetic or chemical means, incapable of metabolizing thymidine; and, (iii) by demonstrating conformity of the transport data to an integrated rate equation derived for a simple, carrier-mediated system. The results indicate that thymidine is transported into mammalian cells by a functionally symmetrical, non-concentrative system for which the carrier : substrate dissociation constant ranges from about 100 μM in Chinese hamster ovary cells, to 230 μM in Novikoff hepatoma cells. In all cell lines investigated, the velocity of transport was sufficient to nearly completely equilibrate low concentrations of thymidine across the membrane within 15 s. Temperature dependence of transport velocity and substrate : carrier dissociation were continuous (EA = 18.3 kcal/mol, ΔH0′ = 9.3 kcal/mol, respectively), and showed no evidence of abrupt transitions. Several natural and artificial nucleosides and nucleic acid based inhibited influx of radiolabeled thymidine, apparently by competing with thymidine for the transport carrier.

Hexose transport in novikoff rat hepatoma cells a simple carrier with directional symmetry, but variable relative mobilities of loaded and empty carrier

AbstractThe kinetics of transport of the non-metabolizable hexose, 3-O-methyl-d-glucose, have been measured in Novikoff rat hepatoma cells by both zero-trans entry and equilibrium exchange procedures. Transport conformed to a simple carrier model which operates symmetrically with respect to direction, but with greater mobility of the loaded than of the empty carrier. Although a complete kinetic description of the transporter can, in theory, be obtained by application of integrated equations describing the time course of substrate equilibrium across the membrane beginning from the zero-trans situation, statistical analysis of hypothetical data indicated that directional asymmetry or differential mobilities of loaded and empty carrier cannot be discerned reliably from such data alone. The difference in mobility of loaded and empty carrier, apparent in a comparison of zero-trans entry and exchange data, ranged from 1.5–7-fold in different batches of cells. It is concluded that the magnitude of the difference is not an inherent property of the transporter, but is determined physiologically, and may be involved in regulation of hexose transport.

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

AbstractThe 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 (Kd = 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.

Regular paperPurine and pyrimidine transport and permeation in human erythrocytes

AbstractTime courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 μM in suspensions of human erythrocytes at 25 or 30°C. At concentrations above 25 μM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the ifnlux and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 μM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations 1 μM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.

Mobility of nucleoside transporter of human erythrocytes differs greatly when loaded with different nucleosides

AbstractTime courses of transmembrane equilibration of 2-chloroadenosine, 2′-deoxyadenosine, 3′-deoxyadenosine, cytidine and 2′-deoxycytidine were measured by rapid kinetic techniques in human erythrocytes under equilibrium exchange and zero-trans conditions. The kinetic parameters for transport were computed by fitting appropriate integrated rate equations to the data pooled for seven concentrations and compared to the kinetic parameters for uridine, adenosine, thymidine and formycin B transport determined previously for human erythrocytes under comparable experimental conditions. The transport of all nucleosides conformed to the simple carrier model and was directionally symmetric. The Michaelis-Menten constants for equilibrium exchange (Kee) ranged from 22 μM for 2-chloroadenosine to about 4 mM for cytidine and the maximum velocities (Vee) differed in a similar manner, so that the first-order rate constants (Vee/Kee) were similar for all nucleosides. The kinetic parameters for 2′-deoxyadenosine transport were similar to those for adenosine transport, whereas the lack of the 3′-OH group greatly reduced the affinity of 3t́-deoxyadenosine (cordycepin) for the carrier, 2′,3′-Dideoxynucleosides were transported < 1% as efficiently as 2′- and 3t́-deoxynucleosides. Thus, the 2′- and 3′-OH groups play an important role in nucleoside transport. The mobility of the carrier when loaded with pyrimidine nucleosides (reflected by Vee was 5–10-times greater than that of the empty carrier, whereas the mobility of th adenosine-loaded or 2′-deoxyadenosine-loaded carrier was about equal to that of the empty carrier. Loading the carrier with 2-chloroadenosine or 3′-deoxyadenosine actually decreased its mobility. Thus, the differential mobility of the loaded and empty carrier differs greatly with the nucleoside substrate. The mobility of the loaded carrier as well as Kee increased with a decrease in lipid solubility of the nucleoside substrate, but the relationship was complex.

Thymidine transport in cultured mammalian cells. Kinetic analysis, temperature dependence and specificity of the transport system

AbstractThe transport of thymidine has been characterized kinetically and thermodynamically in Novikoff rat hepatoma cells grown in culture and, less extensively, in mouse L cells, Chinese hamster ovary cells, P388 murine leukemia cells and HeLa cells. That the characterizations pertained to the transport system per se was ensured, (i) by employing recently developed methods for rapid sampling of cell/substrate mixtures in order to follow isotope movements within a few seconds after initial exposure of cells to substrate; (ii) by utilizing cells rendered, by genetic or chemical means, incapable of metabolizing thymidine; and, (iii) by demonstrating conformity of the transport data to an integrated rate equation derived for a simple, carrier-mediated system. The results indicate that thymidine is transported into mammalian cells by a functionally symmetrical, non-concentrative system for which the carrier : substrate dissociation constant ranges from about 100 μM in Chinese hamster ovary cells, to 230 μM in Novikoff hepatoma cells. In all cell lines investigated, the velocity of transport was sufficient to nearly completely equilibrate low concentrations of thymidine across the membrane within 15 s. Temperature dependence of transport velocity and substrate : carrier dissociation were continuous (EA = 18.3 kcal/mol, ΔH0′ = 9.3 kcal/mol, respectively), and showed no evidence of abrupt transitions. Several natural and artificial nucleosides and nucleic acid based inhibited influx of radiolabeled thymidine, apparently by competing with thymidine for the transport carrier.

Hexose transport in novikoff rat hepatoma cells a simple carrier with directional symmetry, but variable relative mobilities of loaded and empty carrier

AbstractThe kinetics of transport of the non-metabolizable hexose, 3-O-methyl-d-glucose, have been measured in Novikoff rat hepatoma cells by both zero-trans entry and equilibrium exchange procedures. Transport conformed to a simple carrier model which operates symmetrically with respect to direction, but with greater mobility of the loaded than of the empty carrier. Although a complete kinetic description of the transporter can, in theory, be obtained by application of integrated equations describing the time course of substrate equilibrium across the membrane beginning from the zero-trans situation, statistical analysis of hypothetical data indicated that directional asymmetry or differential mobilities of loaded and empty carrier cannot be discerned reliably from such data alone. The difference in mobility of loaded and empty carrier, apparent in a comparison of zero-trans entry and exchange data, ranged from 1.5–7-fold in different batches of cells. It is concluded that the magnitude of the difference is not an inherent property of the transporter, but is determined physiologically, and may be involved in regulation of hexose transport.

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

AbstractThe 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 (Kd = 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.

Regular paperPurine and pyrimidine transport and permeation in human erythrocytes

AbstractTime courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 μM in suspensions of human erythrocytes at 25 or 30°C. At concentrations above 25 μM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the ifnlux and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 μM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations 1 μM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.

Mobility of nucleoside transporter of human erythrocytes differs greatly when loaded with different nucleosides

AbstractTime courses of transmembrane equilibration of 2-chloroadenosine, 2′-deoxyadenosine, 3′-deoxyadenosine, cytidine and 2′-deoxycytidine were measured by rapid kinetic techniques in human erythrocytes under equilibrium exchange and zero-trans conditions. The kinetic parameters for transport were computed by fitting appropriate integrated rate equations to the data pooled for seven concentrations and compared to the kinetic parameters for uridine, adenosine, thymidine and formycin B transport determined previously for human erythrocytes under comparable experimental conditions. The transport of all nucleosides conformed to the simple carrier model and was directionally symmetric. The Michaelis-Menten constants for equilibrium exchange (Kee) ranged from 22 μM for 2-chloroadenosine to about 4 mM for cytidine and the maximum velocities (Vee) differed in a similar manner, so that the first-order rate constants (Vee/Kee) were similar for all nucleosides. The kinetic parameters for 2′-deoxyadenosine transport were similar to those for adenosine transport, whereas the lack of the 3′-OH group greatly reduced the affinity of 3t́-deoxyadenosine (cordycepin) for the carrier, 2′,3′-Dideoxynucleosides were transported < 1% as efficiently as 2′- and 3t́-deoxynucleosides. Thus, the 2′- and 3′-OH groups play an important role in nucleoside transport. The mobility of the carrier when loaded with pyrimidine nucleosides (reflected by Vee was 5–10-times greater than that of the empty carrier, whereas the mobility of th adenosine-loaded or 2′-deoxyadenosine-loaded carrier was about equal to that of the empty carrier. Loading the carrier with 2-chloroadenosine or 3′-deoxyadenosine actually decreased its mobility. Thus, the differential mobility of the loaded and empty carrier differs greatly with the nucleoside substrate. The mobility of the loaded carrier as well as Kee increased with a decrease in lipid solubility of the nucleoside substrate, but the relationship was complex.

Research paper2-Deoxycoformycin inhibition of intracellular phosphorylation of adenosine in Novikoff rat hepatoma cells

AbstractTreatment of cultured wild type and azaguanine-resistant Novikoff rat hepatoma cells with 0.1 to 100 μM 2-deoxycoformycin resulted in an inhibition of more than 50 per cent of the incorporation of 100 μM [8-3H]adenosine into intracellular ATP and nucleic acids. In wild type cells, part of the effect resulted from an inhibition of adenosine deamination of deoxycoformycin, with a consequent decrease of incorporation of radioactivity from adenosine via inosine → hypoxanthine → IMP. This pathway, however, was blocked in azaguanine-resistant cells because of the lack of hypoxanthine/guanine phosphoribosyltransferase. The inhibition of adenosine incorporation by deoxycoformycin in these cells was not mediated at the level of adenosine transport or phosphorylation of AMP. We conclude, therefore, that the intracellular phosphorylation of adenosine is impaired in deoxycoformycin-treated cells. There was a close correlation between inhibition of adenosine deamination and adenosine incorporation, both with respect to effective concentrations of deoxycoformycin and to irreversibility of the inhibition. In addition, intracellular concentrations of adenosine above 1–5 μM were found to strongly inhibit the phosphorylation of adenosine in sity, reflecting substrate inhibition of adenosine kinase. The results indicate that the inhibition of adenosine phosphorylation in deoxycoformycin-treated cells was caused by the accumulation of free adenosine in these cells due to adenosine deaminase inhibition.

Chapter 16 A Rapid-Mixing Technique to Measure Transport in Suspended Animal Cells: Applications to Nucleoside Transport in Novikpff Rat Hepatoma Cells

Publisher SummaryThe chapter discusses techniques, which permit an operational separation of transport and metabolism. This separation can be achieved by genetic, chemical, or kinetic manipulation, or a combination thereof. The transport of various compounds across mammalian cell membranes is frequently found to occur with a rapidity which necessitates collecting data at intervals of a few seconds. By means of a dual-syringe device, suspended cells can be mixed nearly instantaneously with radioactively labeled substrate and separated from the substrate again within seconds by centrifugation into silicone oil. Depending on the cell-substrate system under investigation, initial transport velocities may be either measured directly or calculated from the time course with which equilibrium across the membrane is attained. With nonmetabolizing systems, the dual-syringe apparatus is adaptable to a variety of experimental protocols-zero-trans, equilibrium exchange, and infinite-cis—which in combination make possible a thorough kinetic characterization of a transport system.

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