Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells

Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.     

Thymidylate synthetase catalyzed exchange of tritiumfrom [5-3H]-2'-deoxyuridylate for protons of water.

Thymidylate synthetase catalyzes an exchange of tritium of [5-3H]dUMP for protons of water in the absence of CH2-H4folate. The turnover number for this reaction is some 45,000-fold lower than that of dTMP formation and Km is 1.2 X 10(-5) M, similar to the dissociation constant of the enzyme-dUMP complex determined by equilibrium dialysis. The presence of 4 mM folate has no effect on Vmax but results in a decrease in the Km of dUMP to a value close to that in the normal enzymic reaction. The exchange reaction provides definitive evidence that the enzymic reaction involves attack of a nucleophile of the enzyme on the 6 position of dUMP to provide a 5,6-dihydro-dUMP intermediate which is covalently bound to the enzyme. Stereochemical considerations of the exchange reaction require proposal of a partial reaction which is not completely sterospecific or a complex reaction in which protons of water are handled with complete stereospecificity in a fashion similar to the one carbon unit of the normal enzymic reaction.     

The incorporation of deoxyuridine monophosphate into DNA increases the sister-chromatid exchange yield

The effect of a treatment with 5-fluoro-2'-deoxyuridine (FdUrd) in combination with 2'-deoxyuridine (dUrd) on cell proliferation, incorporation of DNA precursors into DNA and sister-chromatid exchanges (SCEs) has been analyzed in Allium cepa meristem cells. FdUrd in the range 10(-9)-5 X 10(-7) M produced a dose- and time-dependent decrease in the amount of cells in mitosis. This inhibitory effect could be reversed by 70-80% in short-term (6 h) experiments, by exogenously supplied dUrd at a concentration of 10(-4) M. However, at the highest FdUrd dose tested (10(-7) M), 10(-4) M dUrd could not reverse the FdUrd effect in long-term experiments (20 h, about one cell cycle interval), as shown by analyzing the kinetics of synchronous cell populations. DNA extracted from cells pulsed with [6-3H]dUrd in the presence of FdUrd and 6-amino-uracil (6-AU), an inhibitor of uracil-DNA glycosylase, contained a small amount of label (at least 3% of the total radioactivity incorporated into DNA) in the form of [6-3H]dUMP. Thus, we conclude that, under our experimental conditions, exogenously supplied dUrd may be metabolized intracellularly to 2'-deoxyuridine triphosphate (dUTP) and that this deoxynucleotide may eventually be mis-incorporated into DNA. As far as the formation of SCEs is concerned, analysis of second division chromosomes showed that 2'-deoxyuridine monophosphate (dUMP) residues present in newly-synthesized DNA strands are probably not relevant to SCE formation. However, by analyzing SCE levels in third division chromosomes of cells treated with FdUrd and dUrd during their second cycle, we have scored a 6-fold increase in the reciprocal SCE level which demonstrates that the replication of a dUMP-containing DNA template leads to a higher SCE yield.     

The turnover of deoxyuridine triphosphate during the HeLa cell cycle

The synthesis and breakdown of deoxyuridine triphosphate (dUTP) was studied to determine whether a dUTP pool is present at any stage of the HeLa cell cycle. Although cell extracts were found to be capable of phosphorylating dUMP to dUTP, only minimal quantities of intracellular dUMP, dUDP or dUTP could be detected. When thymidylate synthetase was blocked with FUdR the dUMP pool increased but no substantial increase in dUDP or dUTP was seen. A powerful and specific dUTP nucleotidohydrolase (dUTPase, EC3.6.1.23) which hydrolyses dUTP to dUMP and PPi was detected. The activity of this enzyme as well as that of the dUTP synthesizing enzymes was low in G1, rose through S and G2 and reached a maximum just prior to cell division. Pulsing experiments with [5-³H]UdR and [¹⁴C]TdR suggest that the size of the dUTP pool is 1% of the dTTP pool.     

Methionine synthase assay based on coupling with thymidylate synthase reaction

Methionine synthase reaction may be coupled with thymidylate synthase-catalysed tritium release from [5-3H]dUMP via non-enzymatic reaction of formaldehyde with tetrahydrofolate. A convenient and sensitive assay of methionine synthase activity, based on this principle, is described.     

Thymidylate synthase activity in the development of Hymenolepis diminuta

Extracts of the tapeworm, Hymenolepis diminuta, catalyse N5,10-methylene-tetrahydrofolate-dependent release of tritium from [5-3H]dUMP, indicating the presence of thymidylate synthase. The enzyme activity was found in immature, mature and gravid proglottids, as well as in immature and mature oncospheres. The reaction showed pH optimum at 7.5. Its Michaelis constants were approximately 2 and 15 microM for dUMP and (+/-), L-N5,10-methylenetetrahydrofolate, respectively. Incubation of the tapeworm extracts with 5-F-[3H]dUMP and N5,10-methylenetetrahydrofolate resulted in formation of a labelled complex, separable under conditions of SDS polyacrylamide electrophoresis (mol. wt. of approx. 34,000), corresponding to thymidylate synthase subunit. Results of gel filtration of the above complex, under nondenaturing conditions, pointed to a dimeric structure of the enzyme.     

An analysis of myo-[3H]inositol trisphosphates found in myo-[3H]inositol prelabelled avian erythrocytes.

Evidence is presented to show that acid extracts of avian erythrocytes prelabelled for 24-48 h with myo-[3H]inositol contain the following myo-[3H]inositol trisphosphates (expressed as a percentage of total myo-[3H]inositol trisphosphates extracted): 36% myo-[3H]inositol 1,4,5-trisphosphate; 33.7% myo-[3H]inositol 1,3,4-trisphosphate; 13% myo-[3H]inositol 3,4,5-trisphosphate; 9.7% myo-[3H]inositol 3,4,6-trisphosphate; 4.4% myo-[3H]inositol 1,4,6-trisphosphate and 3.3% myo-[3H]inositol 1,3,6-trisphosphate. The only phosphatidyl-myo-[3H]inositol bisphosphate that could be detected in [3H]Ins-prelabelled avian erythrocytes was phosphatidyl-myo-[3H]inositol 4,5-bisphosphate. Cellular myo-[3H]inositol 3,4,5-trisphosphate may be synthesized by dephosphorylation of myo-[3H]inositol 3,4,5,6-tetrakisphosphate. D- and L-myo-[3H]inositol 1,4,6-trisphosphate and D- and L-myo-[3H]inositol 1,3,6-trisphosphate may be dephosphorylation products of myo-[3H]inositol 1,3,4,6-tetrakisphosphate.     

Thymidylate synthase inhibition in cells with arrested DNA synthesis is not due to an allosteric interaction in the replitase complex

Activity of thymidylate synthase was measured in situ in leukemia cells by tritium release from [5-3H]dUrd. Aphidicolin, an inhibitor of DNA polymerase alpha, but not thymidylate synthase, caused a time dependent inhibition of the enzyme when added to the cells after [5-3H]dUrd. Cells treated with hydroxyurea and aphidicolin in sequence before addition of [5-3H]dUrd had a high initial thymidylate synthase activity that decreased with time. This pattern indicates that thymidylate synthase activity is linked to DNA synthesis; however, its inhibition by drugs that inhibit DNA synthesis may be due to accumulation of thymidine nucleotide(s), rather than to an allosteric interaction in the replitase complex.     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

Synthesis and Evaluation of 6‐Aza‐2′‐deoxyuridine Monophosphate Analogs as Inhibitors of Thymidylate Synthases,and as Substrates or Inhibitors of Thymidine Monophosphate Kinase in Mycobacterium tuberculosis

A series of 5‐substituted analogs of 6‐aza‐2′‐deoxyuridine 5′‐monophosphate, 6‐aza‐dUMP, has been synthesized and evaluated as potential inhibitors of the two mycobacterial thymidylate synthases (i.e., a flavin‐dependent thymidylate synthase, ThyX, and a classical thymidylate synthase, ThyA). Replacement of C(6) of the natural substrate dUMP by a N‐atom in 6‐aza‐dUMP 1a led to a derivative with weak ThyX inhibitory activity (33% inhibition at 50 μM ). Introduction of alkyl and aryl groups at C(5) of 1a resulted in complete loss of inhibitory activity, whereas the attachment of a 3‐(octanamido)prop‐1‐ynyl side chain in derivative 3 retained the weak level of mycobacterial ThyX inhibition (40% inhibition at 50 μM ). None of the synthesized derivatives displayed any significant inhibitory activity against mycobacterial ThyA. The compounds have also been evaluated as potential inhibitors of mycobacterial thymidine monophosphate kinase (TMPKmt). None of the derivatives showed any significant TMPKmt inhibition. However, replacement of C(6) of the natural substrate (dTMP) by a N‐atom furnished 6‐aza‐dTMP ( 1b ), which still was recognized as a substrate by TMPKmt.     

Biochemical evidence for the existence of thymidylate synthase in the obligate intracellular parasite Chlamydia trachomatis.

Since eucaryotic cell-derived thymidine or thymidine nucleotides are not incorporated into Chlamydia trachomatis DNA, we hypothesized that C. trachomatis must obtain dTTP for DNA synthesis by converting dUMP to dTMP. In most cells, this reaction is catalyzed by thymidylate synthase (TS) and requires 5,10-methylenetetrahydrofolate as a cofactor. We used C. trachomatis serovar L2 and a mutant CHO K1 cell line with a genetic deficiency in folate metabolism as a host for chlamydial growth. This cell line lacks a functional dihydrofolate reductase (DHFR) gene and, as a result, is unable to carry out de novo synthesis of dTTP. C. trachomatis inclusions form normally when DHFR- cells are starved for thymidine 24 h prior to and during the course of infection. When [6-3H]uridine is used as a precursor to label C. trachomatis-infected CHO DHFR- cells, radiolabel is readily incorporated into chlamydia-specific DNA. When DNA from [6-3H]uridine-labelled infected cultures is acid hydrolyzed and subjected to high-performance liquid chromatography analysis, radiolabel is detected in thymine and cytosine nucleobases. By using the DHFR- cell line as a host and [5-3H]uridine as a precursor, we could monitor intracellular C. trachomatis TS activity simply by following the formation of tritiated water. There is a good correlation between in situ TS activity and DNA synthesis activity during the chlamydial growth cycle. In addition, both C. trachomatis-specific DNA synthesis and 3H2O release are inhibited by exogenously added 5-fluorouridine but not by 5-fluorodeoxyuridine. Finally, we demonstrated in vitro TS activity in crude extracts prepared from highly purified C. trachomatis reticulate bodies. The activity is dependent on the presence of methylenetetrahydrofolic acid and can be inhibited with 5-fluoro-dUMP. Taken together, these results indicate that C. trachomatis contains a TS for the synthesis of dTMP.     

Differential effect of detergents on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral-type benzodiazepine-binding sites

The present study demonstrates a differential effect of various detergent treatments on [3H]Ro 5-4864 and [3H]PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 (0.0125%) caused a decrease of about 70% in [3H]Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on [3H]PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in [3H]Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in [3H]Ro 5-4864 and [3H]PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in [3H]Ro 5-4864 binding, while [3H]PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin. These results may further support the assumption that Ro 5-4864 and PK 11195 are agonist and antagonist, respectively, of PBS and interact with two different conformations or domains in the peripheral-type benzodiazepine binding site molecule.     

Thymidylate synthetase-deficient mouse FM3A mammary carcinoma cell line as a tool for studying the thymidine salvage pathway and the incorporation of thymidine analogues into host cell DNA.

A thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-), auxotrophic for thymidine (dThd), proved extremely useful for studying the dependence of cell growth on the exogenous supply of dThd, the relation between cell growth and DNA synthesis, and the ability of a series of 25 5-substituted 2'-deoxyuridines (dUrd) to substitute for dThd in sustaining cell growth. FM3A/TS-cells did not proliferate unless dThd was supplied to the cell culture medium. The 5-halogenated dUrd derivatives 5-chloro-dUrd, 5-bromo-dUrd and 5-iodo-d Urd also sustained FM3A/TS- cell growth. The extents of incorporation of [methyl-3H]dThd and 5-iodo-[6-3H]dUrd into DNA were closely correlated with their stimulatory effects on FM3A/TS- cell growth. This suggests that the stimulatory effects of the dUrd analogues on the growth rate of FM3A/TS- cells may be considered as evidence for their incorporation into host cell DNA. Based on this premise it is postulated that, in addition to 5-chloro-dUrd, 5-bromo-dUrd, 5-iodo-dUrd and dThd itself, the following dThd analogues are also incorporated into FM3A/TS- cell DNA (in order of the extent to which they are incorporated): 5-hydroxy-dUrd greater than 5-propynyloxy-dUrd greater than 5-ethyl-dUrd greater than 5-ethynyl-dUrd approximately 5-vinyl-dUrd. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to dissociate the de novo and salvage pathways of dTMP biosynthesis and to distinguish those dUrd analogues that are incorporated into DNA from those that are not.     

Heterogeneity of [3H]inositol 1,4,5-trisphosphate binding sites in adrenal-cortical membranes. Characterization and validation of a radioreceptor assay.

1. The characterization of a radioreceptor assay for determining Ins(1,4,5)P3 concentration in tissue extracts is described which utilizes the binding of [3H]Ins(1,4,5)P3 to an adrenal-cortex membrane fraction. 2. Analysis of [3H]Ins(1,4,5)P3 binding by isotope dilution demonstrated an apparent single population of binding sites (KD 3.65 +/- 0.18 nM, Bmax. 872 +/- 70 fmol/mg of protein). Specific binding of [3H]Ins(1,4,5)P3 was enhanced at alkaline pH values (maximum at pH 8.5), with complete loss of specific binding at pH less than 6. These binding sites displayed strict stereo- and positional specificity for Ins(1,4,5)P3, with L-Ins(1,4,5)P3, Ins(1,3,4)P3 and DL-Ins(1,3,4,5)P4 causing 50% displacement of specific [3H]Ins(1,4,5)P3 binding (IC50 values) at concentrations of 14 +/- 3 microM, 3.0 +/- 0.3 microM and 0.53 +/- 0.03 microM respectively. 3. Kinetic analysis of binding data, however, revealed a high-affinity [3H]Ins(1,4,5)P3 binding site (KD 0.052 nM) in addition to the lower-affinity site (KD 2.53 nM) already demonstrated in displacement studies. 4. It is shown that the presence of the high-affinity site can be exploited to increase the sensitivity of the [3H]Ins(1,4,5)P3 radioreceptor assay, allowing accurate detection of 20 fmol of Ins(1,4,5)P3 in 300 microliters of tissue extract. 5. Further validation of the specificity of the above assay for Ins(1,4,5)P3 was provided by incubating tissue extracts with either a 5-phosphatase or 3-kinase preparation. It was shown that identical loss occurred of both Ins(1,4,5)P3 mass and [3H]Ins(1,4,5)P3, added to parallel incubations. 6. The ability of the assay to measure basal and agonist-stimulated increases in Ins(1,4,5)P3 concentration has been demonstrated with rat cerebral cortex and bovine tracheal smooth-muscle slices and a range of cultured and isolated cell preparations.     

A [3H]lysine-containing synthetic peptide substrate for human protocollagen lysyl hydroxylase

A tridecapeptide containing tritium-labelled lysine and corresponding closely to residues 98 to 110 of the alpha chain of type I collagen was synthesized by the solid-phase method. Gly-Leu-Hyp-Gly-Nle-[4,5-3H]Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was used as a substrate of human protocollagen lysyl hydroxylase (peptidyllysine, 2-oxoglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4) obtained from dermal fibroblasts. L-[4,5-3H]Lysine was converted to N alpha-t-butyloxycarbonyl-N epsilon-o-chlorobenzyloxycarbonyl [3H]lysine which was incorporated during stepwise synthesis of the peptide. The chemical and radiochemical purities and specific activity of the completed peptide were characterized. A non-radiolabelled analogue of the peptide inhibited the hydroxylation of [3H]lysine-containing protocollagen by human lysyl hydroxylase, indicating that the synthetic peptide interacted with the enzyme. The peptide containing [3H]lysine was a substrate for lysyl hydroxylase and permitted direct measurement of enzyme activity in relatively crude cell extracts by a tritium-release assay. Extracts of cultured fibroblasts from a patient with an autosomal recessive pattern of inheritance for Ehlers-Danlos syndrome type VI had activities for tritium release from either the radiolabelled synthetic peptide or from [3H]lysine-containing protocollagen that were only 30% of those from control cells. These data indicate that a stable, well-defined synthetic peptide containing [3H]lysine is a useful substrate for studies of genetically variant lysyl hydroxylase from cultured human cells.     

Selective inhibition of [3H]nitrendipine binding to brain and cardiac membranes by bacitracin

The effects of bacitracin were investigated on [3H]nitrendipine binding to rat brain and cardiac membranes in a low ionic strength (5 mM Tris-HCl) buffer. Bacitracin inhibited [3H]nitrendipine binding to rat brain and cardiac membranes with IC50 values of 400 +/- 100 and 4600 +/- 400 micrograms/mL, respectively. Scatchard analysis in brain membranes revealed that bacitracin inhibited [3H]nitrendipine binding primarily by reducing the Bmax but also by producing a small increase in the Kd. In brain membranes, Na+ (100 mM) and Ca2+ (2 mM) reduced the potency of bacitracin to inhibit [3H]nitrendipine binding by approximately sixfold with IC50 values of 2600 +/- 300 and 2100 +/- 400 micrograms/mL observed for bacitracin in the presence of 100 mM Na+ and 2 mM Ca2+, respectively. The EC50 values for the effects of Na+ and Ca2+ were 800 +/- 200 microM and 25 +/- 5 mM. K+, Mg2+, choline, and increasing the assay buffer of Tris-HCl to 50 mM also decreased the inhibition of [3H]nitrendipine binding by bacitracin. These results suggest that bacitracin specifically modulates [3H]nitrendipine binding in a cation-dependent manner and that brain and cardiac dihydropyridine binding sites are either biochemically different or exist in a different membrane environment.     

Equilibrium binding characteristics of [3H]thiomuscimol.

The equilibrium binding characteristics of the tritiated GABAA agonist, 5-aminomethyl-3-isothiazolol (thiomuscimol) are described. Using the filtration technique to separate bound- from free-ligand, [3H]thiomuscimol was shown to bind to the GABA(A) receptor site(s) in a saturable manner with a Kd value of 28+/-6.0 nM and a Bmax value of 50+/-4.0 fmol/mg original tissue. In parallel binding experiments, the Kd and Bmax values for [3H]muscimol were determined to be 5.4+/-2.8 nM and 82+/-11 fmol/mg original tissue, respectively. In binding assays using the centrifugation technique, Kd and Bmax values for [3H]thiomuscimol were found to be 116+/-22 nM and 154 13 fmol/mg original tissue, respectively, whereas a Kd value of 16+/-1.8 nM and a Bmax value of 155+/-8.0 fmol/mg original tissue were determined for [3H]muscimol. In comparative inhibition studies using the GABA(A) antagonist SR 95531 and a series of specific GABAA agonists, the binding sites for [3H]thiomuscimol and [3H]muscimol were shown to exhibit similar pharmacological profiles. Autoradiographic studies disclosed similar regional distribution of [3H]thiomuscimol and [3H]muscimol binding sites in rat brain. Highest densities of binding sites were detected in cortex, hippocampus, and cerebellum, whereas low densities were measured in the midbrain structures of rat cortex. In conclusion, the equilibrium GABA(A) receptor binding characteristics of [3H]thiomuscimol are very similar to those of [3H]muscimol.     

Identification of specific [3H]adenine-binding sites in rat brain membranes

We recently reported that adenine acts as a neurotrophic factor independent of adenosine or P2 receptors in cultured Purkinje cells [Watanabe S. et al. (2003) J. Neurosci. Res. 74, 754-759], suggesting the presence of specific receptors for adenine in the brain. In this study, the characterization of adenine-binding activity in the rat brain was performed to further characterize the receptor-like adenine-binding sites. Specific binding sites for [(3)H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [(3)H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 x 10(5) M(-1) min(-1) and 0.118 +/- 0.045 min(-1), respectively. A single binding site for [(3)H]adenine with a K (D) of 157.1 +/- 20.8 nM and a B (max) of 16.3 +/- 1.1 pmol/mg protein (n = 6) was demonstrated in saturation experiments. A displacement study involving various related compounds showed that the [(3)H]adenine binding was highly specific for adenine. It was also found that [(3)H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [(3)H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [(3)H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes.     

Underestimation of D-glucose phosphorylation as measured by 3H2O production from D-[2-3H]glucose

In rat pancreatic islets, tumoral islet cells (RINm5F line), parotid gland, and in human erythrocytes, but not in rat hepatocytes, the production of 3H2O from D-[2-3H]glucose is 20-30% lower than from D-[5-3H]glucose. This coincides with the production of tritiated lactic acid from D-[2-3H]glucose and may be attributable to an intramolecular hydrogen transfer in the phosphoglucoisomerase reaction. It is concluded that the production of 3H2O from D-[2-3H]glucose is not a reliable tool to assess the total rate of hexose phosphorylation.     

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.