Independent characterization of thymidine transport and subsequent metabolism in Hymenolepis diminuta--II. Purification and preliminary analysis of thymidine kinase

1. An affinity column for the purification of thymidine kinase (TK) from the cestode Hymenolepis diminuta is described. Using an epoxy-activated Sepharose 6B affinity column containing thymidine as a ligand, a 698-fold purification of thymidine kinase was obtained. 2. Thymidine kinase eluted from this affinity column was partially characterized as having an apparent Km value of 3.94 microM thymidine. This value is very similar to those observed in mammalian systems. 3. Thymidine kinase appears to be an extremely active and ubiquitous enzyme, whose primary function is to rapidly phosphorylate incoming thymidine and thus "trap" it for the cell's use, reducing efflux to a minimum. 4. The apparent Km for TK is two orders of magnitude lower than the Kt for thymidine transport. Thus, theories postulating that long-term (2 min) uptake kinetics for thymidine actually represent subsequent metabolism must look further along the thymidine phosphorylating pathway, beyond TK and its very active role.     

Effect of x-rays on the intracellular transport of thymidine in human lymphocytes stimulated by phytohemagglutinins

The effect of X-irradiation on thymidine transport in human lymphocytes PHA stimulated was investigated. Mediated transport sistem, the predominant mechanism at low extracellular concentration of thymidine (less than 10(-7) M) in the medium is highly radiosensitive. The transport sistem was damaged considerably by high doses of X-rays (at least until 10 Krad); the decrease of thymidine uptake was a function of the time of incubation after irradiation. It suggest that repair mechanisms are not involved at high doses of X-rays within 120 minutes of incubation.     

Characterization of a phosphate transport system in human fibroblast lysosomes.

The uptake of [32P]KH2PO4 by Percoll-purified human fibroblast lysosomes at pH 7.0 was investigated to determine if lysosomes contain a transport system recognizing phosphate. Lysosomal phosphate uptake was linear for the first 2 min, attained a steady state by 8-10 min at 37 degrees C, and was not Na+ or K+ dependent. Upon entering lysosomes, [32P]phosphate was rapidly metabolized to trichloroacetic acid-soluble and trichloroacetic acid-insoluble products. After 1-min incubations, 50% of the radioactivity recovered from lysosomes was in the form of inorganic phosphate; and after a 2.5-min incubation, 27% of the radioactivity was recovered as inorganic phosphate. When lysosomes are loaded with radioactivity by incubation with 0.03 mM [32P]KH2PO4 for 25 min and then washed at 4 degrees C, lysosomes fail to release the accumulated radioactivity during a subsequent incubation at 37 degrees C. Lysosomal phosphate uptake gave linear Arrhenius plots (Q10 = 1.8) and was inversely proportional to medium osmolarity. Phosphate uptake was maximal at pH 5-6, half-maximal at pH 7.1, with little transport activity at pH greater than 8, suggesting that the transport system recognizes the monobasic form of phosphate. Lysosomal phosphate uptake is saturable, displaying a Km of 5 microM at pH 7.0 and 37 degrees C. High specificity for phosphate is demonstrated since large concentrations of Na2SO4, NaHCO3, KCl, NaCl, 5'-AMP, or the anion transport inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonate, have no effect on lysosomal phosphate transport. In contrast, the phosphate analog, arsenate, strongly inhibits lysosomal phosphate uptake in a competitive manner with a Ki of 7 microM. Pyridoxal phosphate, CTP, adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), and glucose 6-phosphate were found to be noncompetitive inhibitors of lysosomal phosphate uptake displaying Ki values of 80-250 microM. When lysosomes are incubated with [gamma-32P]ATP, the lysosomal membrane ATPase hydrolyzes the ATP to form inorganic phosphate which then enters lysosomes by this lysosomal phosphate transport route.     

Uptake of thymidine into isolated rat hepatocytes. Evidence for two transport systems

Thymidine transport was studied in isolated rat hepatocytes. In these cells no phosphorylation of the substrate by thymidine kinase occurred subsequent to transport. Results from studies of the concentration-dependent uptake of thymidine indicated two transport systems with about 80-fold differences in their kinetic constants. These systems were denoted as high affinity [Km = 5.3 micron, V = 0.47 pmol/(10(6) cells X s)] and low affinity systems [Km = 480 micron, V = 37.6 pmol/(10(6) cells X s)]. From intracellular to extracellular distribution ratios of [3H]thymidine it could be concluded that the uptake by the high affinity system was a concentrative process while the transport by the low affinity system was non-concentrative. The uptake of [3H]-thymidine by the high affinity system could only be inhibited by unlabeled thymidine. In contrast, all other nucleosides tested (uridine, 2'-deoxycytidine, and 2'-deoxyguanosine) were equally effective in inhibiting the low affinity system competitively. The results would suggest that in hepatocytes lacking phosphorylation by thymidine kinase, thymidine is taken up by a high and a low affinity system working in tandem. The high affinity system seems to be an active transport process with narrow substrate specificity. Thymidine uptake by the low affinity system is a facilitated diffusion process. This system is considered to be a common transport route for nucleosides of different structures.     

Reversal by trypsin of the inhibition of active transport by colicin E1.

The time course for inhibition of proline transport and irreversible loss of cell viability after treatment with colicin E1 was measured as a function of temperature between 13 and 33 degrees C, using a thermostatted flow dialysis system. Complete inhibition of proline transport at 33 and 13 degrees C occurred in 0.5 min and 3 to 5 min, respectively, after addition of colicin E1 at an effective multiplicity of about 4. At these times, the fractional cell survival, assayed by dilution directly from the flow dialysis vessel into trypsin, ranged from 35 to 80%, with viability always greater than 50% at the lower incubation temperatures. Further studies were carried out at 15 degrees C. Complete inhibition of proline transport, which required 2 to 3 min, occurred much more rapidly at 15 degrees C than did the decay of trypsin rescue, which required 10 to 15 min to reach a survival level of 10 to 20%. The direct addition of trypsin to the flow dialysis vessel, after an addition of colicin E1 that caused complete inhibition of proline or glutamine transport, resulted in restoration of net transport. The restored level was typically about 40% of the control rate, and was very similar to the fractional cell viability measured after incubation in trypsin in the same vessel. It is concluded that trypsin can restore active transport to a significant fraction of a cell population in which transport has been initially inhibited by colicin E1.     

Methionine transport in Yersinia pestis.

Yersinia pestis TJW, an avirulent wild-type strain, requires phenylalanine and methionine for growth. It was of interest to examine and define the methionine transport system because of this requirement. The methionine system showed saturation kinetics with a Km for transport of approximately 9 times 10(-7) M. After 8 s of methionine transport, essentially all of the methionine label appeared in S-adenosyl-L-methionine (SAM) as detected in ethanol extracts. Small amounts of free methionine was detected intracellularly after 1 min of transport. Addition of glucose increased significantly the amount of intracellular methionine at 1 min. A series of SAM metabolic products was detected after 90 s to 5 min of transport including: 5'-thiomethyladenosine, homoserine lactone, S-adenosyl homoserine, and a fluorescent methyl receptor compound. Results from assays for SAM synthetase in spheroplast fractions showed a small (16%) but significant portion of synthetase associated with the membrane. However, most of the enzyme activity was associated with the cytoplasmic fraction. Methionine transport was characterized by a high degree of stereospecificity. No competition occurred from structurally unrelated amino acids. Although uptake was inhibited by uncoupling and sulfhydryl reagents, no efflux was observed. Results using energy inhibitors on unstarved and starved cells showed that respiratory inhibitors such as potassium cyanide (KCN) and amytal were most effective, and that arsenate was least effective. KCN plus arsenate completely blocked utilization of energy derived from glucose, and KCN completely blocked utilization of energy deived from D-lactate. The data indicate that methionine transport in Y. pestis is linked to the trapping of methionine in SAM. The results further suggest that this transport system can be classified as a permease-bound system where transport is coupled to an energized membrane state and to respiration.     

Training does not protect against exhaustive exercise-induced lactate transport capacity alterations

The effects of endurance training on lactate transport capacity remain controversial. This study examined whether endurance training 1) alters lactate transport capacity, 2) can protect against exhaustive exercise-induced lactate transport alteration, and 3) can modify heart and oxidative muscle monocarboxylate transporter 1 (MCT1) content. Forty male Wistar rats were divided into control (C), trained (T), exhaustively exercised (E), and trained and exercised (TE) groups. Rats in the T and TE groups ran on a treadmill (1 h/day, 5 days/wk at 25 m/min, 10% incline) for 5 wk; C and E were familiarized with the exercise task for 5 min/day. Before being killed, E and TE rats underwent exhaustive exercise (25 m/min, 10% grade), which lasted 80 and 204 min, respectively (P < 0.05). Although lactate transport measurements (zero-trans) did not differ between groups C and T, both E and TE groups presented an apparent loss of protein saturation properties. In the trained groups, MCT1 content increased in soleus (+28% for T and +26% for TE; P < 0.05) and heart muscle (+36% for T and +33% for TE; P < 0.05). Moreover, despite the metabolic adaptations typically observed after endurance training, we also noted increased lipid peroxidation byproducts after exhaustive exercise. We concluded that 1) endurance training does not alter lactate transport capacity, 2) exhaustive exercise-induced lactate transport alteration is not prevented by training despite increased MCT1 content, and 3) exercise-induced oxidative stress may enhance the passive diffusion responsible for the apparent loss of saturation properties, possibly masking lactate transport regulation.     

3'-azido-3'-deoxythymidine. An unusual nucleoside analogue that permeates the membrane of human erythrocytes and lymphocytes by nonfacilitated diffusion

The demonstrated in vitro and in vivo activity of 3'-azido-3'-deoxythymidine (N3dThd) against the infectivity and the cytopathic effect of human immunodeficiency virus has prompted an investigation of the mechanism by which this nucleoside analogue permeates the cell membrane. As with the transport of thymidine, the influx of N3dThd into human erythrocytes and lymphocytes was nonconcentrative during short incubation times (less than 5 min) which did not allow significant metabolism of this nucleoside. However, in contrast with thymidine transport, the initial velocity of N3dThd influx was strictly a linear function of nucleoside concentration (0.5-10 mM), without evidence of saturability; insensitive to micromolar concentrations of potent inhibitors of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep); insensitive to a 1000-fold excess of other nucleosides (thymidine, uridine, 2-chloroadenosine); and relatively insensitive to temperature, with Q10 values (37-27 degrees C) of 1.4 and 2.7 for N3dThd and thymidine, respectively, determined in erythrocytes. Although the above results indicate that N3dThd permeates the cell membrane chiefly by nonfacilitated diffusion and not via the nucleoside transporter, millimolar concentrations of this nucleoside analogue were observed to inhibit both zero-trans influx of thymidine and efflux of thymidine from [3H]thymidine-loaded erythrocytes. The partition coefficients (1-octanol:0.1 M sodium phosphate, pH 7.0) of N3dThd and thymidine were determined to be 1.26 and 0.064, respectively. The unusual ability of N3dThd to diffuse across cell membranes independently of the nucleoside transport system may be attributed to the considerable lipophilicity imparted to this molecule by the replacement of the 3'-hydroxyl group of thymidine with an azido moiety.     

Monosaccharide uptake by erythrocytes of the embryonic and adult chicken

Rates of monosaccharide uptake by adult and 10-18 day old embryonic chicken erythrocytes were quantitated. The rate of carrier-mediated, stereospecific transport decreased 28% from day 10 to day 14 of incubation and was unchanged thereafter. At no time, however, did the rate of carrier-mediated transport by embryonic erythrocytes differ significantly from that of the adult cells. The rate of transfer by simple diffusion was 3-5 fold faster in embryonic than in adult erythrocytes. Uptake by simple diffusion decreased slightly as the embryo developed. Chronic hyperoxic incubation (70% O2) had little influence on total monosaccharide uptake by embryonic erythrocytes.     

Inhibitors of membrane transport system for organic anions block fura-2 excretion from PC12 and N2A cells.

The neuroblastoma-like cell line N2A and the pheochromocytoma-like cell line PC12 excrete about 20-25% of the intracellular fluorescent Ca2+ indicator fura-2 during 10 min of incubation at 37 degrees C. The drug probenecid, known to inhibit membrane systems for the transport of organic anions [Cunningham, Israili & Dayton (1981) Clin. Pharmacol. 6, 135-151], inhibited fura-2 excretion in both cell types. However, probenecid also had untoward effects on intracellular Ca2+ homeostasis in N2A and PC12 cells. We therefore tested the drug sulphinpyrazone, another known inhibitor of organic-anion transport systems. Sulphinpyrazone fully inhibited excretion of fura-2 at 250 microM, a concentration one order of magnitude lower than that of probenecid. At this concentration and for incubation times up to 20 min, sulphinpyrazone had no untoward effects on cell viability and metabolic functions. Fura-2 was also loaded into the cytoplasm of N2A cells by permeabilization of the plasma membrane with extracellular ATP. In this case as well, the dye was rapidly released from the cells and the efflux was blocked by sulphinpyrazone. These findings suggest that N2A and PC12 cells possess a membrane system for the transport of the free-acid form of fura-2. This transport system is probably responsible for the excretion of fura-2 from these cells. Incubation of N2A and PC12 cells with sulphinpyrazone may help overcome problems arising in the investigation of [Ca2+]i homeostasis in these cell types.     

Dependence of phosphate transport in yeast on glycolytic substrates

Preincubation of baker’s yeast (wild strain, respiration-deficient mutant and a low-phosphorus culture) with glucose, trehalose, and other metabolic sugars increases the subsequent uptake of inorganic phosphate 3 – 5 times. The K_t is reduced by the preincubation from 3.5 to 1.6 mm. The process involves primarily the production of glycolytic energy sources (supression by iodoacetamide, no effect of antimycin or dicyclohexylcarbodiimide, negligible effect of ethanol, or respiratory mutation). The low-phosphorus yeast takes up phosphate anions about 10–20 times faster than the high-phosphorus (normal) culture. The stimulation is also accompanied by some (apparently nonessential) protein synthesis and has a half-time of 35 min; its decay has a t_0.5 of 12 min but affects only less than one-half of the stimulated capacity.     

Differences of affinity and energetics of the active transport systems for amino acids in Chang liver cells.

The plasma membrane of Chang liver cells was shown to have at least two distinct active transport systems, one with preferential affinity for glycine and one for leucine. The uptakes of glycine and leucine were specificially inhibited by Me-AIB and b-BCH, respectively. The uptake of glycine decreased remarkably within 10 min on incubation with DNP (2 mM), KCN (5 mM), and malonate (20 mM) under aerobic conditions, along with a decrease of cellular ATP concentration to as low as 1/4 of normal, while the uptake of leucine was not depressed under these conditions. Leucine uptake was, however, greatly reduced within 10 min on incubation with DNP plus ICH2CONH2 (5 mM), when the cellular ATP was estimated at about 0.066 mM. The active transport of leucine, but not that of glycine, was accompanied by further acidification of the intracellular fluid, which was lower in pH than the extracellular fluid by approximately 0.3 unit without addition of amino acid to the medium.     

Glycoprotein transport in the surface mucous cells of the rat stomach

The intracellular transport of glycoproteins pulse-labeled in vitro with tritiated leucine and galactose in the surface mucous lining cells (SMC) of the fundus of the rat stomach was studied by electron microscope autoradiography. The SMC survive for several hours in pieces of the fundus incubated in a bicarbonate-buffered medium. The SMC have a normal ultrastructure for at least 4 h of incubation. Kinetic activity is normal for at least 5 h, as demonstrated by the normal nuclear incorporation of tritiated thymidine; The SMC incorporate labeled leucine and galactose at normal rates up to 4 h and 6 h, respectively. In contrast to the SMC, the cells of the gastric glands show signs of degeneration within 1 h after the start of incubation. In the SMC the secretory protein forms a smaller part of the total protein synthesized than in other secretory cells studied. The intracellular tranpsort of the leucine-labeled moiety of the glycoproteins follows the normal pathway. The RER loses 35% of its transportable labeled protein within 30 min. The Golgi complex is maximally labeled at 40 min and the mucous granules after 120 min. Galactose is attached to the glycoproteins mainly in the Golgi complex. Glycoproteins are not secreted within 2 h after synthesis of their protein moiety.     

A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine.

The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition.     

The inhibition of hexose transport by permeant and impermeant sulfhydryl agents in rat adipocytes

The importance of exofacial sulfhydryl groups for hexose transport and its regulation was studied by comparing the effects of plasma membrane-permeant maleimide (N-ethylmaleimide) to an impermeant maleimide (glutathione-maleimide I) on 3-O-methylglucose transport into isolated rat adipocytes. The impermeant nature of glutathione-maleimide was confirmed by the finding that after a 15-min incubation, concentrations as high as 10 mM had no effect on intracellular glutathione content, while 1.7 mM N-ethylmaleimide decreased intracellular glutathione by 61%. Although N-ethylmaleimide appeared to be a more potent inhibitor of transport below 5 mM and at incubation times of less than 5 min, neither agent at concentrations which did not cause significant cell breakage inhibited basal transport rates more than 60-70%. The inhibition of transport by both agents was unaffected by extensive washing, suggesting a possible covalent interaction with the carrier. Preincubation with p-chloromercuribenzenesulfonic acid protected against the transport inhibition induced by both agents. However, only the transport inhibition induced by glutathione-maleimide was prevented by preincubation with D-glucose (50 mM) and maltose (50 mM). Transport in cells pretreated with insulin was inhibited by both agents to a similar extent as basal transport. However, treatment of cells with the maleimides before insulin caused a greater degree of inhibition. Thus, the insulin-induced increase in transport was inhibited half-maximally by 1 mM glutathione-maleimide. These results show that exofacial sulfhydryl groups, perhaps on the hexose-binding site of the carrier, are important for both the function and regulation of hexose transport.     

Effects of lower alcohols on potassium transport and microsomal adenosine-triphosphatase activity of rat cerebral cortex

1. Slices of rat cerebral cortex, incubated anaerobically at 37 degrees , lost K(+) from an initial concentration of 102m-equiv./kg. to a concentration of 57m-equiv./kg. after 10min. On subsequent aerobic incubation they regained K(+) rapidly at a rate that varied with the K(+) concentration of the medium. 2. Lower aliphatic alcohols, present at equal thermodynamic activity, produced approximately equal degrees of inhibition of K(+) uptake during the aerobic incubation. This inhibition was reduced by an increase in K(+) content of the medium. Ethanol did not affect the rate of K(+) loss during anaerobic incubation. 3. Li(+), in concentrations of 1-10mm, also inhibited K(+) uptake by brain-cortex slices, the degree of inhibition varying with the Li(+) concentration. Ouabain also inhibited K(+) uptake. 4. The same series of alcohols, at equal thermodynamic activity, produced comparable degrees of inhibition of Na(+),K(+),Mg(2+)-stimulated adenosine-triphosphatase activity in brain microsomes. 5. It is suggested that inhibition of cation transport is an important, but not a primary, mechanism in the production of central nervous depression by alcohols and other substances.     

Evidence for two sites of inhibition of photosynthetic electron transport by dibromothymoquinone

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 μm. At concentrations of about 10 μm, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of p-phenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and $\text{KCN}\text{Hg}$-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 μm dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.     

Kinetics of methionine transport and metabolism by Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense

Methionine is an essential amino acid for both prokaryotic and eukaryotic organisms; however, little is known concerning its utilization in African trypanosomes, protozoa of the Trypanosoma brucei group. This study explored the Michaelis-Menten kinetic constants for transport and pool formation as well as metabolic utilization of methionine by two divergent strains of African trypanosomes, Trypanosoma brucei brucei (a veterinary pathogen), highly sensitive to trypanocidal agents, and Trypanosoma brucei rhodesiense (a human pathogenic isolate), highly refractory to trypanocidal arsenicals. The Michaelis-Menten constants derived by Hanes-Woolf analysis for transport of methionine for T. b. brucei and T. b. rhodesiense, respectively, were as follows: K(M) values, 1. 15 and 1.75 mM; V(max) values, 3.97 x 10(-5) and 4.86 x 10(-5) mol/L/min. Very similar values were obtained by Lineweaver-Burk analysis (K(M), 0.25 and 1.0 mM; V(max), 1 x 10(-5) and 2.0 x 10(-5) mol/L/min, T. b. brucei and T. b. rhodesiense, respectively). Cooperativity analyses by Hill (log-log) plot gave Hill coefficients (n) of 6 and 2 for T. b. brucei and T. b. rhodesiense, respectively. Cytosolic accumulation of methionine after 10-min incubation with 25 mM exogenous methionine was 1.8-fold greater in T. b. rhodesiense than T. b. brucei (2.1 vs 1.1 mM, respectively). In African trypanosomes as in their mammalian host, S-adenosylmethionine (AdoMet) is the major product of methionine metabolism. Accumulation of AdoMet was measured by HPLC analysis of cytosolic extracts incubated in the presence of increasing cytosolic methionine. In trypanosomes incubated for 10 min with saturating methionine, both organisms accumulated similar amounts of AdoMet (approximately 23 microM), but the level of trans-sulfuration products (cystathionine and cysteine) in T. b. rhodesiense was double that of T. b. brucei. Methionine incorporation during protein synthesis in T. b. brucei was 2.5 times that of T. b. rhodesiense. These results further confirm our belief that the major pathways of methionine utilization, for polyamine synthesis, protein transmethylation and the trans-sulfuration pathway, are excellent targets for chemotherapeutic intervention against African trypanosomes.     

Mechanisms of iron transport into rat erythroid cells

Three mechanisms of iron uptake by rat erythroid cells were identified, two with non-transferrin-bound iron (NTBI) and one with transferrin-bound iron (Fe-Tf). Uptake of NTBI occurred by a high affinity mechanism (K(m) approximately 0.1 microM). Activity of the high affinity mechanism was maximal in sucrose solution and of the low affinity mechanism in KCl solution. Both were inhibited by NaCl and by certain ion transport inhibitors, but they differed in their sensitivity to the various inhibitors. Fe-Tf uptake was also of high affinity (K(m) 0.1 microM). All the transport mechanisms show higher activity in reticulocytes than in mature erythrocytes, and all could provide iron for heme synthesis in reticulocytes. The results demonstrate certain conditions which should be followed in order to study high affinity transport of NTBI. These include use of a low packed cell volume in the incubation mixture, low iron concentrations (0.01-1.0 microM), short incubation times (up to 20 min), and low osmolality (approximately 200 mOsm/kg) during incubation with the NTBI and subsequent washing of the cells.     

Loss of a termolabile thymidine kinase activity in bromodioxyuridine resistant (transport deficient, kinase postive) haploid cultured frog cells.

Haploid frog cell strain ICR B20 is resistant to 10^?4 M bromodeoxyuridine through its failure to express a thymidine-specific saturable transport reaction, although it continues to express thymidine kinase (EC 2.7.1.75) at levels comparable to that of the sensitive parental strain ICR 2A. However, the thymidine kinases of these two lines are not entirely equivalent: we report here that ICR 2A enzyme appears to contain two fractions of differing thermostability and that ICR B20 expresses only the more thermostable form. Plots of log surviving enzyme activity vs time at 45 °C from ICR 2A are biphasic and may be resolved into a thermolabile component F with a half-life about 5 min and a thermostable component S with a half-life greater than 200 min. Similar plots from ICR B20 are exponential and indicate a half-life greater than 200 min. The fraction of the initial enzyme activity represented by F averages about one-third of the total. If ICR 2A enzyme is heated in the presence of thymidine (0.05 mM), component F is stabilized. However, dialysis against thymidine-free buffer does not make S activity thermolabile. The time course of enzyme reaction in ICR 2A preparations is non-linear and suggests that F activity may decay independently of S; ICR B20 extracts (and extracts of ICR 2A that have previously been heated at 45 °C) give nearly linear reaction rates. These observations suggest that the transport deficiency of ICR B20 is associated with loss of a portion of the total thymidine kinase activity and raise the possibility that the lost fraction of activity may participate in transport.