Nucleoside transport in mammalian cell membranes

Summary Transport of the nucleoside analog cytosine-arabinoside (CAR) in transformed hamster cells in culture has been studied in conditions of minimal metabolic conversion. Uptake (zero-trans in) properties at 20°C over a limited range of CAR concentrations were characterized by aK _m of 350 m and a maximal velocity (V) of 780 m·min^–1 (V/K _m =2.28 min^–1). Equilibrium exchange at 20°C over a wider range of concentrations was best described by a saturable component with aK _m of 500 m and av of 1230 m·min^–1 (V/K _m =2.26 min^–1) and either a saturable component of highK _m or a nonsaturable component ofk=0.3 min^–1. For the saturable component, thev/K _m values were similar in both procedures.CAR transport was inhibited by various metabolizable nucleosides. Uptake of some of these nucleosides was inhibited by CAR. CAR transport and uridine uptake were inhibited in a reversible but partially competitive fashion by high affinity probes like S-(p-nitrobenzyl-6-mercaptoinosine (NBMI) (K _i <0.5nm) and in an irreversible fashion by SH reagents such as N-ethylmaleiimide (NEM). The organomercurialp-hydroxymercuribenzene sulfonate (pMBS) markedly stimulated transport of these nucleosides, but also markedly potentiated the inhibitory effects of either NBMI or NEM. These effects are interpreted either in terms of models which invoke allosteric properties or in terms of two transport systems which display distinct chemical susceptibilities to externally added probes.     

Nucleoside transport in mammalian cell membranes

Organomercurials form stable stoichiometric complexes with thiolated nucleosides. The complexes inhibited uptake of ribonucleosides and cytosine arabinoside (CAR) in various types of normal and transformed cells. The inhibition was competitive and reversible (Ki = 3--6 micrometer). The interaction between complexes and transport system displayed a 1:1 stoichiometry. Chemical factors which contributed to the inhibitory power were evaluated with a series of S-alkylated derivatives and S--Hg--R complexes of mercaptonucleosides. The inhibitory potency was not determined exclusively by the hydrophobic nature of either the S-alkylated or the S--Hg--R moieties. Chemical modification of cells with penetrating and nonpenetrating organomercurials lead to stimulation of nucleoside uptake and to an increase in its susceptibility to inhibition by S--Hg--R complexes or S-aklylated derivatives of mercaptopurine ribosides. The kinetic and chemical data obtained with nucleoside analogs and with chemical modifiers suggested complex features of nucleoside transport systems. Four distinct classes of sites were implied: (i) a substrate binding site susceptible directly to competitive inhibition by organomercurial-mercaptonucleoside complexes, (ii) an additional site susceptible either to S-arylalkylated or S-mercuriated derivatives of 6-mercaptopurine ribosides, (iii) SH-containing modifier sites which stimulate uridine uptake upon binding of organomercurials, and (iv) SH-containing modifier sites which inhibit the function upon binding of organomercurials. From the observation that only SH sites related to stimulation were susceptible to modification by macromolecular-SH modifier probes, some conclusions can be drawn regarding the disposition of the various sites in the cell membrane in general and among membrane components in particular.     

Kinetic characteristics and regulation of hexose transport in a galactokinase-negative Chinese hamster fibroblast cell line: a good model for studies on sugar transport in cultured mammalian cells

We report the kinetic characteristics for D-galactose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose transport in a galactokinase null-allele mutant of a Chinese hamster V79 cell line. GalKl cells exhibited a Km and Vmax for D-galactose, 2-deoxy-D-glucose, and 3-O-methyl-D-glucose transport of 8.6 +/- 2.6 mM and 26.1 +/- 7.2 nmol/mg p/min, 4.1 +/- 1.2 mM and 40.3 +/- 9.5 nmol/mg p/min, and 7.01 +/- .85 mM and 11.6 +/- 4.8 nmol/mg p/30 s, respectively. Nonsaturable hexose uptake was determined using cytochalasin B inhibition of galactose uptake (89.6 +/- 3.7% of galactose uptake was cytochalasin B inhibitable) and L-glucose uptake (7.5% of the galactose uptake). D-Galactose was not metabolized and effluxed rapidly from preloaded cells. The Kls for the inhibition of D-galactose transport were 4.5 +/- 2.5 mM for D-glucose, 7.0 +/- 2.0 mM for 2-deoxy-D-glucose, 6 mM for 2-deoxy-D-galactose and 6.0 +/- 0.6 mM for 3-O-methyl-D-glucose. This indicates the operation of a single common carrier. The hexose transport rate decreased 50-60% after 24 h serum deprivation. Addition of insulin was shown to increase hexose transport (more than twofold) in serum-deprived cells. Hexose transport rates increased substantially in glucose-deprived, D-fructose- or D-galactose-fed cells as compared to glucose-fed cells. Since GalKl does not metabolize galactose, the hexose transport increases induced by feeding cells galactose suggest that carrier interaction with ligand is not a significant factor in transport regulation in GalKl. The kinetic and regulatory characteristics of D-galactose transport in the GalKl cell line indicate that this system is a good model to study sugar transport from a mechanistic and regulatory point of view.     

Inhibition of uridine transport in cultured mammalian cells by theophylline

Theophylline (theobromine, caffeine) reversibly inhibits the incorporation of labeled RNA precursors both in confluent 37 RC and in exponentially growing HeLa cells. As measured in 37 RC after 2 h labeling, 20 mM theophylline reduces the incorporation of [3H]UTP and [14C]uridine into acid-precipitable material to 5% and 9% of the control, respectively. This reduction is paralleled by a comparably lowered incorporation of the same precursors into the acid-soluble pool. The initial rate of incorporation into total cell material is similarly affected by theophylline, the inhibition being of a simple competitive type. Theophylline does not alter the turnover rate of pulse labeled RNA during actinomycin D chase nor does it preclude the utilization of the endogenous pool of nucleoside phosphates. Upto a concentration of 10 mM, it does not inhibit uridine kinase neither in 37 RC nor in HeLa cells. The mentioned inhibitory effects of theophylline cannot be mimicked by exogenously added cyclic AMP. All the data support the conclusion that theophylline inhibits the transport of uridine into the cell.     

Nucleoside transport in human erythrocytes. Nitrobenzylthioinosine binding and uridine transport activities have similar radiation target sizes

Intact human erythrocytes were irradiated in the frozen state with a high-energy electron beam. Nitrobenzylthioinosine-sensitive uridine influx, equilibrium exchange uridine influx and high-affinity nitrobenzylthioinosine binding were inactivated as a simple exponential function of the radiation dose, indicating an in situ target size of 122 000. The results suggest that the nitrobenzylthioinosine-binding site(s) and the permeation site(s) of the transporter are present on the same transporter element.     

Cytogenetic assays of chemical clastogens using mammalian cells in culture.

A protocol is described for cytogenetic assays of chemical mutagens using mammalian cells in vitro. The system employs continuous drug treatment (3 concentrations) for up to 8 h and recovery-cell populations after pulse treatments with a high dose. Both direct fixation (for recording spindle anomalies in anaphase) and colcemid-hypotonic fixation (for reading metaphase chromosome aberrations) are used in order to estimate the effects of an agent as a mitotic poison and as a clastogen respectively. Some DNA intercalating dyes (acridine orange, quinacrine mustard, neutral red) were found to be highly clastogenic whereas others (quinacrine dihydrochloride, 33258 Hoechst) are not.     

Selective modification of cell surface proteins and thymidine transport in hamster cells exposed to cholera toxin

The increased adherence and morphological response which occurs in Chinese hamster ovary cells as a result of exposure to cholera toxin is paralleled by modification in the relative exposure of outer proteins. Mild proteolysis treatment of the cells prelabeled with [³H] glucosamine reveals a markedly different kinetics of release of external glycopeptides as a result of exposure to cholera toxin. Selective alterations in external tyrosyl-rich proteins can also be detected by lactoperoxidase-catalyzed radioiodination. The above modifications are accompanied by a decrease in the rate of thymidine uptake by toxintreated cells.     

The mechanism of interaction between high-affinity probes and the uridine transport system of mammalian cells.

The carrier of uridine transport in hamster cells in culture is highly susceptible to the inhibitory effect of probes like S-benzylated derivatives of mercaptopurine nucleosides. The interaction between the probes and the carrier is competitive and reversible and it takes place at a site different from the substrate binding site. The Ki for the most potent derivative p-nitrobenzyl-6-mercaptoinosine is 0.15 n Molar at 20 degrees C. The effect of the probes is interpreted in terms of conformational change induced on the carrier upon binding of the probe. The carrier assumes distinct conformations depending on whether it is probe-free (form A) or probe bound (form B). Kinetic as well as chemical evidence supports the predictions of the allosteric carrier model. A single component of kinetics is observed either in the absence of inhibitor (Km form A) or at high concentrations of inhibitor (Km form B). A two component kinetics is observed at intermediate concentrations of inhibitor (some carriers in form B and others in form A). The two forms have distinct Km values for uridine: form A50 muMolar and form B 250 muMolar. Two forms have also different susceptibilities to the action of organomercurials: form A is insensitive whereas form B is highly inhibited by the chemical modified of SH groups. The existence of putative allosteric sites in carriers is discussed in terms of modifier sites capable of modulating transport activities as a result of specific membrane-ligand interactions.     

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

The 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 microM in Chinese hamster ovary cells, to 230 microM in Novikoff hepatoma cells. In all cell lines investigated, the velocity of transport was sufficient to nearly completely equilibrate low concentration of thymidine across the membrane membrane within 15 s. Temperature dependence of transport velocity and substrate : carrier dissociation were continuous (EA = 18.3 kcal/mol, delta H0' = 9.3 kcal/mol, respectively), and showed no evidence of abrupt transitions. Several natural and artificial nucleosides and nucleic acid bases inhibited influx of radiolabeled thymidine, apparently by competing with thymidine for the transport carrier.     

Facilitated transport of inosine and uridine in cultured mammalian cells is independent of nucleoside phosphorylases

The zero-trans uptake of uniformly and base-labeled inosine and uridine was measured a 25 degrees C in suspensions of Novikoff rat hepatoma cells, Chinese hamster ovary cells, mouse L cells, mouse S49 lymphoma cells and a purine-nucleoside phosphorylase-deficient subline thereof (NSU-1), and in monolayer culture of mouse 3T3 and L cells. The initial velocities of uptake of both nucleosides were about the same in all cell lines investigated, regardless of the position of the label or of the substrate concentration between 3 and 300 microM or whether or not the cells possessed uridine or purine-nucleoside phosphorylase activity. The kinetic parameters for the facilitated transport of uridine and inosine were also similar in phosphorylase positive and negative cell lines (K = 120--260 microM and V = 6--40 pmol/microliters cell water per s) and the transport activities of the cells exceeded their total phosphorylase activities by at least 10-fold for uridine and 1--2-fold for inosine. Chromatographic fractionation of the intracellular contents and of the culture fluid showed that the free nucleosides appeared intracellularly prior to and more rapidly than their phosphorolysis products. During the initial 20--60 s of uptake of U-14C-labeled nucleosides the rates of intracellular appearance of ribose-1-P and base were about the same. After several minutes of incubation, on the other hand, the main intracellular component was ribose-1-P whereas the base attained a low intracellular steady-state concentration and accumulated in the medium due to exit transport. Other nucleosides, dipyridamole and nitrobenzylthioinosine, specifically inhibited the transport of uridine and inosine, and depressed the intracellular accumulation of ribose-1-P and the formation of base commensurate with that inhibition. The data indicate that the metabolism of inosine and uridine by the various cell lines can be entirely accounted for by the facilitated transport of unmodified nucleoside into the cell followed by intracellular phosphorolysis.     

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

The 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 ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= 18.3}\text{kcal/mol}\text{, ΔH}{}^0\text{′ = 9.3}\text{kcal/mol}$, 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.     

Kinetic studies of adenylyl cyclase of fat cell membranes

Summary The kinetic behavior of the adenylyl cyclase activity associated with fat cell membranes purified by centrifugation on sucrose gradients was studied. Under most of the conditions explored, with either Mn⁺⁺ or Mg⁺⁺ as the divalent cation in the assay mixtures, the time courses of the reaction were not linear. In the absence of modifiers (i.e., basal activity) or in the presence of insulin, the rate tended to decrease with time; on the other hand, with fluoride or GMP-P(NH)P the curves were concave upwards. To simplify analysis of the results, two kinetic components were defined: an initial component corresponding to the transient rate measured between zero time and 1.5 min of assay and a final component corresponding to the transient rate determined between 3 and 5 min.Over the entire range of Mn⁺⁺ concentration explored (0.5 to 6.0mm), the basal initial rates were slightly higher than the final ones. With Mg⁺⁺ in the range between 1.5 and 2.5mm, the final rates were fourfold lower than the initial ones. Higher or lower Mg⁺⁺ concentrations gave velocity ratios equivalent to those observed with Mn⁺⁺.Insulin clearly decreased the final rates at Mn⁺⁺ concentrations up to 2.5mm. With higher concentrations the effects were completely reversed. The effects of insulin on initial rates measured with Mn⁺⁺, or the initial or final rates measured with Mg⁺⁺, were less evident.Stimulation of adenylyl cyclase activity by fluoride was most pronounced on the final rates. In addition, this stimulation was higher with Mg⁺⁺ than with Mn⁺⁺.Isoproterenol stimulation of adenylyl cyclase was negligible in the presence of Mn⁺⁺ (0.5 to 6.0mm). With Mg⁺⁺ (0.5 to 6.0mm), stimulation was more evident on the final rates. *** DIRECT SUPPORT *** A0130063 00002     

Concentrating mammalian cells I. large-scale animal cell culture

Concentrating mammalian cells in the large-scale cell culture serves different purposes: the retention of animal cells in bioreactor systems to increase the usable cell density and, therefore, the unit productivity; the concentration of mammalian cells for their subsequent reuse. This review describes the present status of both types of concentration, compares the different application possibilities with their advantages/disadvantages, and gives a short future outlook for this field of biotechnology.     

Species differences in nucleoside transport. A study of uridine transport and nitrobenzylthioinosine binding by mammalian erythrocytes.

A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.     

Molecular mechanisms of sugar transport across mammalian and microbial cell membranes

Recent DNA cloning studies have revealed the existence of a large family of homologous sugar transporters in both prokaryotic and eukaryotic organisms. The family includes passive transporters typical of mammalian tissues and active, H(+)-linked sugar transporters from bacteria. Each of these transporters characteristically contains two groups of six putative membrane-spanning alpha-helices separated by a large, hydrophilic, cytoplasmic region. Both the N-terminal and C-terminal regions of the sequence are also predicted to be cytoplasmic. Biophysical and other studies on the human erythrocyte glucose transporter, the only member of the family so far isolated in functional form, suggest that the membrane-spanning alpha-helices associate to form a hydrophilic channel or a substrate-binding cleft extending across the membrane. It is likely that the mechanism of substrate translocation involves alternate exposure of the substrate-binding site to each face of the membrane via a conformational change. Studies in progress on the erythrocyte transporter are beginning to identify regions of the protein involved in substrate binding and the conformational change, and should throw light on the mechanism of sugar translocation in the sugar transporter family as a whole.