An Evaluation of Irreversible Inhibition of Synaptosomal High-Affinity Choline Transport by Choline Mustard Aziridinium Ion

Abstract: Choline mustard aziridinium is a potent, irreversible and selective blocker of sodium-dependent, high-affinity transport of choline into rat forebrain synaptosomes; it was found to be 30 times less potent against low-affinity transport of choline. The IC₅₀ value for high-affinity transport was 0.94 μM, compared to 29 μM for low-affinity uptake. The inhibitory action of choline mustard aziridinium ion on high-affinity transport of choline was graded with respect to time; a 12-fold increase in potency was obtained by increasing the inhibitor preincubation times from 1 to 30 min. Low concentrations of choline mustard aziridinium ion could produce significant blockade of choline carriers providing the exposure time was prolonged. The characteristics of the blockade of synaptosomal high-affinity choline transport by choline mustard aziridinium ion also changed depending upon preincubation time. The kinetics of inhibition of high-affinity choline transport by choline mustard aziridinium ion showed apparent competitive inhibition initially, followed by noncompetitive characteristics at longer preincubations with inhibitor. The rate of irreversible inhibition of carriers by this nitrogen mustard analogue would appear to be rapid; the rate constant was determined to be 5 × 10^?2 s^?1for micromolar concentrations of inhibitor. This action may preclude the transport of the mustard analogue into the nerve terminal, although initially some reversible binding with the carrier may result in the translocation of some choline mustard aziridinium ion into the presynaptic ending. The progressive alkylation of high-affinity carriers by the analogue could indicate the presence of excess carrier sites in the presynaptic membrane, or subpopulations of carriers in an inactive state in equilibrium with active carriers. A model is described for the inhibitory action of choline mustard aziridinium ion on synaptosomal high-affinity choline carriers.     

Carrier-mediated inhibition of choline acetyltransferase

Incubation of rat forebrain synaptosomes with choline mustard aziridinium ion in a sodium-rich medium caused a time-dependent inhibition of the high-affinity transport of choline, as well as a significant decrease in intrasynaptosomal choline acetyltransferase activity. In the absence of added sodium choline uptake by a sodium-independent mechanism was also blocked in a time-dependent manner but intrasynaptosomal choline acetyl-transferase activity was unaltered. Neither monoethylcholine nor hemicholinium-3 changed intrasynaptosomal choline acetyl-transferase activity but competitively inhibited the transport of choline. The results indicate that there may be a fraction of choline acetyltransferase that is closely associated with the sodium-dependent high-affinity choline transport system and that this fraction can be irreversibly inhibited by choline mustard aziridinium ion, perhaps indirectly mediated by alkylation of the carrier.     

Choline mustard: an irreversible ligand for use in studies of choline transport mechanisms at the cholinergic nerve terminal

It has been shown in our laboratory that choline mustard aziridinium ion is a potent and irreversible inhibitor of choline transport into rat brain synaptosomes; this compound showed selectivity for the sodium-dependent, high affinity carrier in that it was 30 times more potent as an inhibitor when compared with the effect on sodium-independent, low affinity choline uptake. In the present study, this mustard analogue did not inhibit synaptosomal uptake of 5-hydroxytryptamine, noradrenaline, or gamma-aminobutyric acid, thereby confirming further the specificity of this compound for the choline carrier. Studies of the effect of depolarization of the nerve terminals on the inactivation of choline carriers by choline mustard were performed. It was determined that alkylation of the carrier was significantly increased in nerve endings previously depolarized. The enhancing effect of depolarization on choline transport velocity and on the alkylation of choline carriers by choline mustard was dependent upon the presence of sodium in the external medium. Possible mechanisms for the enhanced inactivation of choline carriers by choline mustard aziridinium ion are proposed, and kinetic interactions of choline mustard with the high affinity choline carrier and with choline acetyltransferase are reviewed and discussed.     

Regulation of Rat Brain Synaptosomal [3H]Hemicholinium-3 Binding and [3H]Choline Transport Sites Following Exposure to Choline Mustard Aziridinium Ion

Abstract: Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the “ground-state” (basal) high-affinity choline transport but only 50% of “ground-state” hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K⁺-stimulated increases in choline transport and of [³H]hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [³H]hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca²⁺ or presence of high Mg²⁺ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg²⁺ levels removed the ability of the mustard to inhibit K⁺-stimulated increases of either [³H]hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [³H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extracellular Ca²⁺.     

Inhibition of choline transport into human erythrocytes by choline mustard aziridinium ion.

Acetylcholine mustard aziridinium ion inhibited the transport of [3H]choline into human erythrocytes. Treatment of the erythrocytes with 1 X 10(-4) M tetraethylpyrophosphate prevented the inhibition of [3H]choline transport by acetylcholine mustard aziridinium ion. Hydrolyzed acetylcholine mustard aziridinium ion inhibited choline transport both in the presence and absence of 1 X 10(-4) M tetraethylpyrophosphate. The product of hydrolysis was equipotent with acetylcholine mustard in its ability to inhibit choline transport; incubation of this product with sodium thiosulfate prevented inhibition of choline transport thereby indicating the presence of an aziridinium ion. The hydrolysis product is likely to be choline mustard aziridinium ion. Results on the efflux of [3H]choline from erythrocytes in the presence of the proposed choline mustard aziridinium ion showed that the mustard moiety was transported into the red cells on the choline carrier. The rate of efflux of [3H]choline produced by choline mustard aziridinium ion was 55% of that produced by the same concentration of choline. It is concluded that acetylcholinesterase (EC 3.1.1.7) of red cells rapidly hydrolyzes acetylcholine mustard aziridinium ion to acetate and choline mustard aziridinium and the latter compound can act as a potent inhibitor of choline transport. This finding would indicate that the hemicholinium-like toxicity of acetylcholine mustard in the mouse is due to the formation of choline mustard aziridinium ion.     

Kinetics of irreversible inhibition of choline transport in synaptosomes by ethylcholine mustard aziridinium

Summary Ethylcholine mustard aziridinium (ECMA) inhibits choline transport in synaptosomes at a half-maximal concentration of about 20 m. The rate of inhibition falls off rapidly after 10 min and the concentration dependency reaches a plateau at about 100 m. The inhibition is not removed by washing the synaptosomes, and choline and hemicholinium-3 protect the carrier against attack by the mustard. Choline efflux, particularly that stimulated by choline in the medium (transactivation) is also inhibited by the aziridinium compound. Similarly choline influx activated by preloaded internal choline is inhibited by ECMA. The mustard can enter the synaptosomes in an active form but most of the carrier is alkylated when facing the outside. Prior depolarization of the synaptosomes causes an increase in the rate of inhibition by ECMA which is proportionally about the same as the increase in choline influx also caused by depolarization. At low ECMA concentrations the rate of inhibition is that of a first-order reaction with the carrier but at high ECMA concentrations the translocation of the carrier to the outward-facing conformation controls the rate of inhibition. Using a model of choline transport with some simplifying assumptions it is possible to estimate the amount of carrier; cholinergic synaptosomes carry about six times the concentration of carrier found in noncholinergic ones. In noncholinergic synaptosomes the carrier faces predominately out, the reverse in cholinergic ones. The rate constant of carrier translocation is increased by combination with choline some six- to sevenfold to about 3.5 min^–1. The rate constant of ECMA attack on the carrier is about 440m ^–1 sec^–1.     

High-affinity transport and phosphorylation of 2-deoxy-D-glucose in synaptosomes

2-Deoxy-d -glucose (2 DG) entered synaptosomes (from rat brain) by a high-affinity, Na⁺-independent glucose transport system with a K_m, of 0.24 mM. 3-O-methyl-glucose, D-glucose, and phloretin were competitive inhibitors of 2-DG transport with K_i's of 7 mM, 64 μM, and 0·75 μM, respectively. Insulin was without effect. 2-DG uptake was also saturable at high substrate concentrations with an apparent low affinity K_m, of 75 mM, where the K_l, for glucose was 17.5 mM. We are not certain whether the rate-limiting step for the low-affinity uptake system is attributable to transport or phosphorylation. However, the high-affinity glucose transport system probably is a special property of neuronal cell membranes and could be useful in helping to distinguish separated neurons from glial cells.     

THE UPTAKE, METABOLISM AND RELEASE OF HOMOCHOLINE: STUDIES WITH RAT BRAIN SYNAPTOSOMES AND CAT SUPERIOR CERVICAL GANGLION

The accumulation of [³H]homocholine (3-trimethylamino-propan-1-01) by isolated synaptosomes prepared from rat brain was resolved kinetically into a high (K_T= 3.0 μM) and a low (K_T= 14.5 μM) affinity system. Although homocholine was not acetylated by solubilized choline acetyltransferase, 64% of the homocholine accumulated by intact synaptosomes via the high affinity uptake process was acetylated. Homocholine was also acetylated in the superior cervical ganglion of the cat, and the amount of acetylhomocholine formed was increased (12-fold) by preganglionic nerve stimulation. In ganglia, acetylhomocholine was available for release by preganglionic nerve impulses, and its release was Ca²⁺-dependent, It is concluded that homocholine can form a cholinergic false transmitter, and that the substrate specificity of choline acetyltransferase in vitro might be different from that in situ.     

Specificity of Ethylcholine Mustard Aziridinium as an Irreversible Inhibitor of Choline Transport in Cholinergic and Noncholinergic Tissue

The sensitivity of choline transport to inhibition by ethylcholine mustard aziridinium (ECMA) was studied in several tissues. Choline transport was found to be inhibited irreversibly by ECMA in guinea pig and rat synaptosomes but not inhibited in erythrocytes or kidney slices. If this finding can be extended to other tissues ECMA sensitivity may provide a simple criterion for identifying the choline carrier associated with cholinergic tissue.     

Studies on the Alkylation of Choline Acetyltransferase by Choline Mustard Aziridinium Ion

Although a potent irreversible inhibitor of high-affinity choline transport in rat brain synaptosomes, choline mustard aziridinium ion (ChM Az) appeared to be a relatively weak inhibitor of choline acetyltransferase (ChAT) in rat brain homogenates, and evidence for irreversible binding of this compound to the enzyme had not been established. Accordingly, the irreversible inactivation of partially purified rat brain ChAT by ChM Az was studied. This compound is a rather weak inhibitor of the enzyme, with 50% inhibition of ChAT activity achieved following 30 min incubation at 37 degrees C with 0.6 mM ChM Az. This result indicates that although ChM Az has affinity for many nucleophiles there was little diluting effect of the inhibitor in the crude brain homogenate which could be attributed to such reactions (50% inhibition caused by 1.8 mM ChM Az following 10 min incubation). Although the initial binding of ChM Az to ChAT may be of a competitive nature, irreversible bond formation resulted. The time-dependent alkylation reaction conformed to pseudo-first-order kinetics with an observed forward rate constant (kobs) of 0.173 min-1; the half-time (t 1/2) for irreversible binding was about 4 min. The irreversible inactivation of ChAT by ChM Az would appear to be slower than the alkylation of high-affinity choline carriers in synaptosomes by this compound, and the relatively weak inhibitory action of ChM Az against either partially purified ChAT or ChAT activity in crude rat brain homogenates is in striking contrast to previous evidence that ChAT in intact synaptosomes was inhibited irreversibly by lower concentrations of the inhibitor.     

Presynaptic effect of the aziridinium ion of acetylcholine mustard (methyl-2-acetoxyethyl-2'-chloroethylamine) on the phrenic nerve--rat diaphragm preparation.

The rat diaphragm has been used to investigate the neuromuscular blocking action of acetylcholine mustard which yields a potent nicotinic agonist, an aziridinium ion, in aqueous medium. Evidence was obtained that the acetylcholine mustard aziridinium ion impaired neuromuscular activity when the phrenic nerve was stimulated and that the ion did not directly inhibit muscle contraction. Impairment of neuromuscular activity was characterized by a latent period and depended both on the concentration of aziridinium ion and the frequency of stimulation of the phrenic nerve. Elevated concentrations of Ca-2+ and choline changed the response of the rat diaphragm to the aziridinium ion, the former increasing the rate of development of neuromuscular block and the latter protecting against neuromuscular block. These results indicated that the aziridinium ion may act either at the site of choline uptake or have an effect on acetylcholine synthesis in the nerve ending and that impairment of neuromuscular transmission in the rat diaphragm involved the availability of acetylcholine. Similar results were obtained with acetylcholine mustard aziridinium ion subjected to alkaline hydrolysis. This substance is thought to be choline mustard aziridinium ion. Although difficult to prove with the rat diaphragm it is possible that acetylcholinesterase of this preparation could hydrolyze acetylcholine mustard aziridinium ion at the neurotransmitter site and the resultant choline mustard aziridinium ion would interfere with the uptake of choline and eventually prevent neuromuscular transmission. This hemicholinium-like hypothesis for the mechanism of action of choline mustard aziridinium ion is compatible with reported date for toxicity of acetylcholine mustard aziridinium ion in the mouse.     

Choline transport specificity in animal cells and tissues

1. Beta carbolines inhibit choline transport in rat brain. 2. The aziridinium ring on the nitrogen of mustard analogs of choline causes irreversible binding to the carrier in rat brain. 3. The uptake system in rat brain is stereoselective, requires a quaternary nitrogen, and prefers analogs with a nitrogen-oxygen distance of about 3.26 A. 4. In mouse brain troxonium derivatives inhibit choline transport. 5. In cuttlefish optic lobes and torpedo electric organ pyrene derivatives potently inhibit choline transport. 6. In guinea pig placenta, the affinity of the choline carrier remains high even when this molecule lacks one or two methyl groups.     

ADENOSINE UPTAKE BY CHOLINERGIC SYNAPTOSOMES FROM TORPEDO ELECTRIC ORGAN

Pure cholinergic synaptosomes prepared from the electric organ of Torpedo are able to take adenosine up efficiently and convert it to ATP. The apparent K_m of the adenosine uptake is 2.4 μM and the V_m: 518 pmol/30′/mg prot. The uptake system exhibits a high specificity towards adenosine, as shown by the lack of competition with analogues. Tubercidin blocks the uptake competitively and dipyridamole is a very potent non competitive inhibitor (K_i= 4 × 10^-8 M). Considering that during nerve activity ATP is released extracellularly and can modulate transmitter release, the physiological significance of adenosine uptake is discussed as a possible mechanism to terminate the ATP action.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Affinity Labelling and Identification of the High-Affinity Choline Carrier from Synaptic Membranes of Torpedo Electromotor Nerve Terminals with [3H]Choline Mustard

The physiological mechanisms regulating activity of the sodium-dependent, high-affinity choline transporter and the molecular events in the translocation process remain unclear; the protein has not been purified or characterized biochemically. In the present study, [3H]choline mustard aziridinium ion [( 3H]ChM Az), a nitrogen mustard analogue of choline, bound irreversibly to presynaptic plasma membranes from Torpedo electric organ in a hemicholinium-sensitive, and sodium-, time-, and temperature-dependent manner. Specific binding of this ligand was greatest when it was incubated with membranes in the presence of sodium at 30 degrees C. Separation of the 3H-labelled membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that most of the radiolabel was associated with a polypeptide of apparent molecular mass of approximately 42,000 daltons; labelling of this species was abolished in membranes incubated with ligand in the presence of HC-3. Two other 3H-labelled polypeptides were detected, with apparent molecular masses of approximately 58,000 and 90,000 daltons; radiolabelling of the former was also HC-3 sensitive. [3H]ChM Az may be a useful affinity ligand in the purification of the choline carrier from cholinergic neurons.     

Characterization of the Irreversible Inhibition of High-Affinity Choline Transport Produced by Hemicholinium Mustard

The inhibition of high-affinity choline transport by hemicholinium mustard (HCM), an alkylating analogue of hemicholinium-3, was examined in rat brain synaptosomes and guinea pig myenteric plexus. In synaptosomes, 50% high-affinity choline transport inhibition occurs with an HCM concentration of 104 nM (4-min incubation). A 10-min preincubation with 10 microM HCM results in essentially complete (greater than 95%) inactivation that persists after washing. Low-affinity choline transport in synaptosomes is unaffected by HCM inhibition at all concentrations examined (1-50 microM). Time course experiments indicate that the maximum irreversible inhibition (58%) seen after a 1-min preincubation with 500 nM HCM decreases to 46% inhibition after a 15-min preincubation; however, analysis of variance reveals that this difference is not significant. HCM inhibition of acetylcholine release from myenteric plexus-longitudinal muscle preparations persists for at least 2 h after removal of drug from the incubation bath; this inactivation can be prevented by coincubation with a high choline concentration during treatment with the mustard. In contrast, inhibition produced by the parent compound hemicholinium-3 is largely reversed by washing in both preparations examined. The observed potency and selectivity of HCM suggest its usefulness as a covalent probe for high-affinity choline transport.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5-Triiodothyronine (T₃) inhibited L-[¹⁴C]leucine uptake into synaptosomes. Inhibition was competitive with a K_i of 3.1 × 10^?5m . Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l -Thyroxine and analogues inhibited the incorporation of l -[¹⁴C] leucine into cerebral synaptosome protein. At 50 μm , the triiodo-compounds were more inhibitory than tetraiodo->3,5-triiodo-l -thyronine >3,3′,5-triiodothyropro-pionic> l -thyroxine >3,5-diiodo-l -tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5-Triiodothyronine had no effect on respiratory control or 2,4-DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [¹⁴C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm -ouabain. 3,3′,5-Triiodothyronine had no effect on synaptosome fraction ATPase or Na-K ATPase. Addition of T₃ induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm ) or cycloheximide (50μg/ml). [¹⁴C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5-triiodothyronine. There was no inhibition of [¹⁴C]proline uptake or incorporation. The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were     

Pre-steady-state uptake of -glucose by the human erythrocyte is inconsistent with a circulating carrier mechanism

Simulation shows that the four-state mobile carrier model for sugar transport in which the asymmetry arises from unequal rate constants of inward and outward translation of the free-carrier and carrier-sugar complex, does not fit with the observed data for pre-steady-state uptake recently obtained by A.G. Lowe and A.R. Walmsley ((1987) Biochim. Biophys. Acta 903, 547–550). The main reason for this discrepancy is that pre-steady-state fluxes are determined mainly by the dissociation constants K_s of glucose and maltose for the external sites, rather than the K_m (zero-trans_oi) of glucose and the K_i of maltose. The data are also inconsistent with other forms of asymmetric carrier but are fairly consistent with a symmetrical carrier with high-affinity sites for -glucose or with a fixed site carrier model.