Nucleotidase Inhibition Research Articles

Trichomonas vaginalis NTPDase inhibited by lycorine modulates the parasite-neutrophil interaction

Lycorine is an Amaryllidaceae alkaloid that presents anti-Trichomonas vaginalis activity. T. vaginalis causes trichomoniasis, the most common non-viral sexually transmitted infection. The modulation of T. vaginalis purinergic signaling through the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase), and ecto-5'-nucleotidase represents new targets for combating the parasite. With this knowledge, the aim of this study was to investigate whether NTPDase and ecto-5'-nucleotidase inhibition by lycorine could lead to extracellular ATP accumulation. Moreover, the lycorine effect on the reactive oxygen species (ROS) production by neutrophils and parasites was evaluated as well as the alkaloid toxicity. The metabolism of purines was assessed by HPLC. ROS production was measured by flow cytometry. Cytotoxicity against epithelial vaginal cells and fibroblasts was tested, as well as the hemolytic effect of lycorine and its in vivo toxicity in Galleria mellonella larvae. Our findings showed that lycorine caused ATP accumulation due to NTPDase inhibition. The alkaloid did not affect the ROS production by T. vaginalis; however, it increased ROS levels in neutrophils incubated with lycorine-treated trophozoites. Lycorine was cytotoxic against vaginal epithelial cells and fibroblasts; conversely, it was not hemolytic neither exhibited toxicity against the in vivo model of G. mellonella larvae. Overall, besides having anti-T. vaginalis activity, lycorine modulates ectonucleotidases and stimulates neutrophils to secrete ROS. This mechanism of action exerted by the alkaloid could enhance the susceptibility of T. vaginalis to host immune cell, contributing to protozoan clearance.

Early exposure to ethanol is able to affect the memory of adult zebrafish: Possible role of adenosine

Ethanol is one of the most widely consumed drugs in the world, and the effects of ethanol during early development include morphological and cognitive problems. The regulation of adenosine levels is essential for the proper function of major neurotransmitter systems in the brain, particularly glutamate and dopamine; thus, the investigation of the relation of adenosine and memory after early ethanol exposure becomes relevant. Embryos of zebrafish were exposed to 1% ethanol during two distinct developmental stages: gastrula/segmentation or pharyngula. The evaluation of memory, morphology, and locomotor parameters was performed when fish were 3 months old. The effect of ecto-5′-nucleotidase and adenosine deaminase inhibition on the consequences of ethanol exposure with regard to memory formation was observed. Morphological evaluation showed decreases in body length and the relative telencephalic and cerebellar areas in ethanol exposed animals. The locomotor parameters evaluated were not affected by ethanol. In the inhibitory avoidance paradigm, ethanol exposure during the gastrula/segmentation and pharyngula stages decreased zebrafish memory retention. When ethanol was given in the pharyngula stage, the inhibition of ecto-5′-nucleotidase in the acquisition phase of memory tests was able to revert the effects of ethanol on the memory of adults. These findings suggest that the increased adenosine levels caused by ethanol could alter the neuromodulation of important components of memory formation, such as neurotransmitters. The adjustment of adenosine levels through ecto-5′-nucleotidase inhibition appears to be effective at restoring normal adenosine levels and the acquisition of memory in animals exposed to ethanol during the pharyngula stage.

A domino reaction of 3-chlorochromones with aminoheterocycles. Synthesis of pyrazolopyridines and benzofuropyridines and their optical and ecto-5'-nucleotidase inhibitory effects.

A new and efficient domino reaction of 3-chlorochromones with electron-rich aminoheterocycles was developed which allows for a convenient synthesis of a variety of pyrazolo[3,4-b]pyridines, pyrrolo[2,3-b]pyridines, pyrido[2,3-d]pyrimidines and benzofuro[3,2-b]pyridines. The products exhibit strong fluorescence. In addition, they exhibit significant ecto-5'-nucleotidase inhibition properties and cytotoxic behavior.

Alteration of Lithium Pharmacology through Manipulation of Phosphoadenosine Phosphate Metabolism

Bisphosphate 3'-nucleotidase (BPNT1 in mammals and Met22/Hal2 in yeast) is one of five members of a family of signaling phosphatases united through a common tertiary structure and inhibition by subtherapeutic doses of the antibipolar drug lithium. Here we report a role for 3'-nucleotidase and its substrate, 3'-phosphoadenosine 5'-phosphate (PAP), in mediating the cellular effects of lithium. Lithium-induced inhibition of growth in yeast cells may be overcome by dose-dependent heterologous expression of human BPNT1. Disruption of the yeast 3'-nucleotidase gene or treatment of cells with lithium results in a >80-fold accumulation of PAP and leads to potent growth inhibition. These data indicate that the accumulation of a 3'-nucleotidase substrate, such as PAP, mediates the toxicity of lithium. To further probe this model we examined the growth inhibitory effects of lithium under conditions in which PAP biosynthetic machinery was concomitantly down-regulated. Disruption of met3 or met14 genes (ATP sulfurylase or phosphosulfate kinase), transcriptional down-regulation of MET3 through methionine addition, or administration of chlorate, a widely used cell-permeable sulfurylase inhibitor, function to reduce lithium-induced intracellular PAP accumulation and lithium toxicity; all of these effects were reversed by heterologous expression of human sulfurylase and kinase. Collectively, our data support a role for 3'-nucleotidase activity and PAP metabolism in aspects of lithium's mechanism of action and provide a platform for development of novel pharmacological modulators aimed at improving therapies for the treatment of bipolar disorder.

α,β-MeATP augments the UTP contraction of rabbit basilar artery

The mechanism underlying the interaction between α,β-methyleneadenosine 5′-triphosphate (α,β-MeATP) and uridine 5′-triphosphate (UTP) was investigated using the basilar artery of a rabbit. UTP induced a concentration-dependent contraction, whereas P2X receptor agonists, such as α,β-MeATP and 2-methylthioadenosine 5′-triphosphate (2-MeSATP), did not induce any contraction up to 100 μM. α,β-MeATP augmented the UTP contraction two-fold, immediately and reversibly. This effect was observed with ectonucleotidase inhibition with 1 mM Ni 2+, the removal of extracellular Ca 2+ or Evans blue. The contractile response to adenosine 5′- O-(3-triphosphate) (ATPγS), a selective agonist for P2Y 4, was augmented by pretreatment with α,β-MeATP also. ATPγS had no additional effect on the UTP contraction fully activated with α,β-MeATP. UTP (100 μM) did not induce an increase in cytosolic Ca 2+ in a rabbit basilar arterial strip; however, in the presence of 1 mM α,β-MeATP, UTP induced a significant increase in cytosolic Ca 2+. These results suggest that α,β-MeATP facilitates the activation by UTP of the P2Y receptor (P2Y 4) of the rabbit basilar artery through mechanisms other than nucleotidase inhibition, and that it does not do so via a P2X receptor.

Oxidative Modification of Rat Liver 5'-Nucleotidase: The Mechanisms for Protection and Re-Activation

The effect of oxidative stress catalysed by transition metals appears to have a critical relevance for the structure and function not only of membrane lipids but also of integral membrane proteins in a complex lipid-protein assembling, and membrane-dependent function. The integral membrane enzyme 5'-nucleotidase is susceptible to Fe 2+ -ion catalysed oxidative modification, and the extent of enzyme inhibition is in inverse relationship (r = -0.820) with lipid peroxidation (MDA) level. This work is also a comparative study about possible effectiveness of different Fe-ion chelators (deferoxamine, Na-citrate, Na-salicylate, ammonium oxalate and EDTA), antioxidants (GSH, GSH/GSH-Px system, Cu, Zn-SOD and mannitol) and metal cations (Mg 2+ and Mn 2+) to protect or restore Fe 2+ -ion induced 5'-nucleotidase inhibition and to supress Fe 2+ -ion enhanced lipid peroxidation. Among the examined chelators it was only deferoxamine and Na-citrate that exerted a fully protective and reactivating ability; among the antioxidants it was only GSH; among the metal cations it was only Mn 2+. The ability to protect or restore 5'-nucleotidase activity and to diminish chain-induced lipid peroxidation is explicable in terms of: metal-binding ability, capacity of taking iron away from a biological molecule, or ability of transferring the damage to itself. After a short incubation period, the iron associated with enzyme or lipid hydroperoxides could be in a labile coordinative linkage, still able to interact with possible ligands or metal cations.

Complex hippocampal responses to ATP: fade due to nucleotidase inhibition and P2-receptor-mediated adenosine release

When ATP or the related stable analogue, βγ-imidoATP, were applied to rat hippocampal slices showing population spikes larger than 5 mV peak-to-peak amplitude, a depression of spike size was obtained, which showed a marked fade during the 10-min period of superfusion. The inhibitory responses were prevented by adenosine deaminase or 8-phenyltheophylline. Adenosine responses showed no fade. αβ-MethyleneADP enhanced the fade, while suramin at 50 μm prevented the early component of the responses. The results suggest that in slices with large population spikes, inhibitory responses to nucleotides are partly due to their conversion to adenosine, and partly due to the activation of P2 receptors which trigger the release of endogenous adenosine.

Developmental differences in myocardial protection in response to 5′-nucleotidase inhibition

Age-related differences in the activity of 5'-nucleotidase, an enzyme responsible for conversion of high-energy phosphates to their the diffusible precursors, may help to explain age-related differences in tolerance of global myocardial ischemia. Postischemic function and high-energy phosphate content were measured in the hearts of rabbits 7 to 10 days old (neonate), 30 to 40 days old (1 month), and 6 to 12 months old (adult). Hearts in each age group were subjected to 60 minutes of ischemia at 34 degrees C either with no cardioplegia, with unmodified St. Thomas' Hospital cardioplegic solution, or with St. Thomas' Hospital cardioplegic solution with pentoxifylline, a 5'-nucleotidase inhibitor. These groups were compared with one another and with control hearts that were continuously perfused for 1 hour. In adults, addition of pentoxifylline to St. Thomas' Hospital cardioplegic solution restored adenosine triphosphate and total nondiffusible nucleotide levels to control values and improved recovery of cardiac output and developed pressure compared with results with unmodified St. Thomas' Hospital cardioplegic solution. In contrast, biochemical and functional parameters in neonatal hearts were not affected by either unmodified St. Thomas' Hospital cardioplegic solution cardioplegia or St. Thomas' Hospital cardioplegic solution with pentoxifylline. Functional recovery in neonatal hearts subjected to unprotected ischemia was superior to that in the older age groups. In 1-month-old hearts, St. Thomas' Hospital cardioplegia improved recovery compared with recovery after unprotected ischemia, but no incremental improvement in function or high-energy stores was seen with addition of pentoxifylline. The lack of effect of pentoxifylline on neonatal hearts suggest that there is a relative deficiency of 5'-nucleotidase in this age group. This may contribute to the improved functional recovery observed in unprotected hearts. Furthermore, addition of pentoxifylline to adult hearts appears to confer the benefits of low 5'-nucleotidase activity occurring naturally in the neonate.

Adenosine and adenine nucleotides are independently released from both the nerve terminals and the muscle fibres upon electrical stimulation of the innervated skeletal muscle of the frog

The independent release of adenosine and adenine nucleotides upon electrical stimulation was studied in the innervated sartorius muscle of the frog after blockade of the extracellular catabolism of adenosine monophosphate (AMP) through exo-AMP deaminase and ecto-5'-nucleotidase. Nerve stimulation (30 min, 0.2Hz) induced the release of both adenosine (19 +/- 3 pmol) and adenine nucleotides (101 +/- 7 pmol). Experiments performed in the presence of tubocurarine (5 microM) to prevent purine release due to nerve-evoked muscle twitching, or under direct stimulation of the muscle in low calcium solutions to prevent pre-synaptic release of purines, showed that there was an evoked release of adenosine and adenine nucleotides both from the nerve endings and from the twitching muscle fibres. Removal of ecto-5'-nucleotidase inhibition shows that the catabolism of adenine nucleotides released during stimulation contributes in about 50% to the amount of endogenous extracellular adenosine. When only one of the enzymes catabolizing AMP (ecto-5'-nucleotidase or exo-AMP deaminase) was inhibited, the evoked release of adenine nucleotides was undetectable, suggesting that each enzyme is able to catabolize all the AMP formed from adenine nucleotides released upon stimulation. It is concluded that the concentration of endogenous extracellular adenosine is under the control of the relative activities of exo-AMP deaminase and ecto-5'-nucleotidase.

Further studies of the mechanism(s) of polyunsaturated-fatty-acid-mediated increases in intracellular cAMP formation in N1E-115 neuroblastoma cells.

Following earlier observations that increasing the polyunsaturated fatty-acid (PUFA) content of N1E-115 neuroblastoma cells elevated basal and adenosine (Ado)-stimulated intracellular cyclic AMP (cAMP) formation, we carried out studies to determine the mechanism(s) by which PUFA exerted their modulatory effects. Basal increases in cAMP in the PUFA-enriched (PUFA+) cells were evident with short (60 sec) exposure to a phosphodiesterase inhibitor (Ro 20-1724), and increased to a maximum at 20 min; they were not observed in the absence of Ro 20-1724. Forskolin-stimulated cAMP formation in the presence of the Ro compound was 2- to 3-fold higher in the PUFA+ cells. Basal elevations in cAMP were reduced by approximately 70% by exposing the PUFA+ cells to Ado deaminase (ADA) or to an Ado antagonist, and were further increased by inhibiting ADA, which suggested that they could be producing endogenous Ado that activated stimulatory Ado receptors. However, this did not appear to involve PUFA-mediated stimulation of 5'-nucleotidase activity or inhibition of [3H]Ado uptake. Overall, the results of this study indicated that multiple mechanisms are involved in PUFA modulation of cAMP formation.

Myocardial adenosine formation during hypoxia: Effects of ecto-5′-nucleotidase inhibition

Release of adenosine and AMP into epicardial fluid and coronary venous effluent of isovolumic guinea-pig hearts was examined during normoxic (95% O 2) and hypoxic (30% O 2) perfusion with and without the ecto-5′-nucleotidase inhibitor α,β-methylene adenosine diphosphate (AOPCP) ∗ ∗ Abbreviations: AOPCP is α,β-methylene-adenosine diphosphate. . Normoxic epicardial and venous adenosine levels were 221 ± 27 and 67 ± 11 n m, respectively, in untreated hearts. During 15 min of hypoxia, epicardial and venous adenosine levels increased in a phasic manner, reaching maximal values of 498 ± 32 and 441 ± 43 n m, respectively, during the initial 5 min of hypoxia. Epicardial and venous adenosine levels then declined slightly during the subsequent 10 min to 332 ± 33 and 224 ± 34 n m, respectively. Infusion of 50 μ m AOPCP significantly reduced venous adenosine levels during normoxia (< 50% of control), but was without effect on normoxic epicardial adenosine. Epicardial and venous adenosine levels increased during hypoxia with AOPCP but the increases were lower than those for untreated hypoxic hearts. Epicardial and venous adenosine levels recovered to baseline levels following 30 min of reoxygenation in both groups. Epicardial and venous AMP levels were elevated by AOPCP treatment during normoxia and hypoxia. Coronary vascular resistance decreased during hypoxia but the decline in resistance was less in AOPCP treated hearts. It is concluded that whereas basal interstitial adenosine levels appear to be independent of ecto-5′-nucleotidase activity, the hypoxic increase in interstitial adenosine is partially derived from an AOPCP sensitive ecto-5′-nucleotidase. Venous adenosine appears to be significantly dependent on ecto-5′-nucleotidase activity during normoxia and hypoxia. The data are also consistent with a role for endogenous adenosine in regulating coronary vascular resistance during hypoxia.

Enhanced myocardial protection during global ischemia with 5’-nucleotidase inhibitors

Depletion of adenosine triphosphate precursors, such as myocardial adenosine, during global ischemia results in poor postischemic adenosine triphosphate repletion and functional recovery. Neonatal hearts may be more resistant to this deleterious effect of ischemia, because they are characterized by low 5'-nucleotidase activity, which may result in higher sustained endogenous myocardial adenosine triphosphate precursor levels during ischemia. Adult hearts, however, have high levels of 5'-nucleotidase activity leading to depleted precursors during ischemia and poor postischemic functional recovery. Augmenting myocardial adenosine exogenously during ischemia in adult hearts has a beneficial effect on recovery. The present study tested if preservation of nucleotide precursors, better adenosine triphosphate repletion, and enhanced postischemic myocardial recovery in adult hearts could be achieved with a "neonatal" strategy. Therefore 5'-nucleotidase inhibitors were administered to isolated, perfused adult rabbit hearts subjected to 120 minutes of ischemia (at 34 degrees C) to determine if this improved functional recovery. Hearts received St. Thomas' Hospital cardioplegic solution (control hearts) or cardioplegic solution containing 5'-nucleotidase inhibitors: pentoxifylline, thioinosine, [s-(p-nitrophenyl)-4-thioinosine], or thioinosine's dimethyl sulfoxide vehicle alone. After ischemia and reperfusion, recovery of systolic function, diastolic function, and myocardial oxygen consumption was significantly better with 5'-nucleotidase inhibition. No changes in coronary flow were noted. We speculate and are pursuing the theory that the mechanism of 5'-nucleotidase inhibition's favorable action is due to preventing the catabolism, transport, and loss of nucleotide precursors during ischemia, maintaining adenosine triphosphate precursor availability.

Inhibition of lymphocyte 5'-nucleotidase by lectins: effects of lectin specificity and cross-linking ability.

5'-Nucleotidase, an integral glycoprotein enzyme of the lymphocyte plasma membrane, is inhibited cooperatively by the lectin concanavalin A. Because divalent succinyl-concanavalin A is a poor enzyme inhibitor, both binding and lectin-induced cross-linking of 5'-nucleotidase may be necessary for inhibition. Succinyl-concanavalin A does not compete with concanavalin A for binding to the enzyme; however, maleyl-concanavalin A, another poor inhibitor, competes effectively with the parent lectin. Thus, maleyl-concanavalin A binds to the same site as concanavalin A but causes little inhibition, whereas succinyl-concanavalin A does not bind to this site. The monovalent lectin from Ricinus communis (RCA-60) is a more effective enzyme inhibitor than the related divalent lectin (RCA-120), and inactivation of the second low-affinity sugar binding site on RCA-60 does not abolish inhibition, suggesting that multivalent cross-linking is not required for 5'-nucleotidase inhibition. Peanut and wheat germ agglutinins do not inhibit the enzyme, whereas lectins from lentil, pea, soybean, Griffonia simplicifolia, and Phaseolus vulgaris inhibit 5'-nucleotidase with various degrees of effectiveness. The only lectin showing strong positive cooperativity in its interaction with 5'-nucleotidase is concanavalin A.

Determination of mitogenic properties and lymphocyte target sites of Dolichos lablab lectin (DLA): Comparative study with concanavalin A and galactose oxidase cell surface receptors

A mannoside-directed lectin has been isolated and purified from the seeds of Dolichos lablab L. by affinity chromatography. We have established that this glycoprotein, which displays high erythroagglutinating activity without blood group specificity, highly activates murine T lymphocytes, and we have described for the first time its mitogenic properties. Although its main properties are close to those of concanavalin A (Con A), the well-known mannoside-directed mitogen devoid of sugar moiety, several differences were found in some of the early events triggered by the two lectins during lymphocyte mitogenic stimulation: higher level of interleukin-2 (IL-2) synthesis, optimal dose for IL-2 synthesis at suboptimal mitogenic concentration, lack of ecto-5′ nucleotidase inhibition, and lack of mitogenic inhibition at high lectin concentration. Because the two lectins did not act on the cell surface in exactly the same way, we have compared their receptors involved in mitogenesis on the plasma membrane of murine lymphocytes. We had previously established that the polyclonal activation of these cells probably occurred through high-molecular-weight receptors (200–230 kDa). Since the mitogenic stimulation of lymphocyte by galactose oxidase (GO), like that of Con A, was inhibited by DLA, we analyzed the cell surface receptors that were common to these three polyclonal mitogens. After labeling the neuraminidase/GO-treated cell surface glycoproteins with NaB 3H 4, we immunoprecipitated the Con A and DLA receptors which are the target of GO mitogenic action. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the precipitates demonstrated that there exist on the lymphocyte cell surface receptors common to the polyclonal mitogens DLA, Con A, and GO. Because Con A and DLA sterically inhibit GO mitogenic stimulation, the common glycoproteins which represent the necessary sites of oxidative mitogenic action are probably those which are involved in DLA and Con A-triggered mitogenesis, despite the different properties of the two lectins. These differences could be explained by the lower molecular weight receptors of the two lectins which are not identical.

Erythrocyte pyrimidine 5'-nucleotidase inhibition by acute lead exposure in neonatal rats.

Inhibition by lead of erythrocyte pyrimidine 5'-nucleotidase (P5N) is thought to contribute to morphological abnormalities observed in red blood cells (RBC) of lead-exposed subjects. However, neither the mechanism of lead inhibition of P5N nor the relationship of this inhibition to blood lead levels attained in exposed subjects is known. In the present investigation, acute in vivo and in vitro lead acetate effects on erythrocyte P5N from 21-day-old rat pups were determined and were related to blood lead concentrations ascertained by atomic absorption spectrophotometry. Acute lead administration to rat pups resulted in a 16% to 21% reduction in erythrocyte P5N, with mean blood lead levels ranging from 77 to 108 micrograms/dl 24 hours later. Inhibition of erythrocyte P5N was linearly related to blood lead level (r = -0.67, P less than 0.05) following acute lead administration. Lead acetate addition to RBC preparations from 21-day-old rats resulted in concentration-dependent P5N inhibition which was comparable to that produced following acute in vivo exposure. The results indicate that acute P5N inhibition in lead-treated neonatal rats is due to noncompetitive P5N inhibition by lead. The inhibition of P5N produced by acute lead treatment is linearly related to blood lead concentrations.

The presence and orientation of ecto-5'-nucleotidase in rat liver lysosomes

Purified rat liver lysosomes contained 5'-nucleotidase activity which was 92 ± 2% [4] latent. This latency was lost in response to a permeant sugar at a similar rate to that of the lysosomal marker enzyme β- N-acetyl-glucosaminidase indicating that the 5'-nucleotidase was genuinely located in the lysosome and not a plasma membrane contaminant. Lysosomal 5'-nucleotidase exhibited the following properties characteristic of ecto-5'-nucleotidase inhibition by specific polyclonal antibodies: binding to a monoclonal antibody; inhibition by 1 mmol l αβ-methylene ADP; immunoreactive subunits of 70 and 38 kDa. Lysosomes in addition contained immunoreactive species of intermediate molecular mass.

The proposed 5′-nucleotidase inhibitor in human leukemic cells is an artefact

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5′-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577–584) mentioned that this phenomenon resulted from the presence of a 5′-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5′-[ 3H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [ 3H]Adenosine derived from 5′-[ 3H]AMP hydrolysis was further transformed into [ 3H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [ 3H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5′-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5′-nucleotidase inhibitor. We did not observe 5′-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.

Regulation of purine biosynthesis in cultured Drosophila melanogaster cells : II/relationships between hypoxanthine-guanine-phosphoribosyl transferase and 5′-nucleotidase

All in vitro cell lines from Drosophila melanogaster do not possess measurable hypoxanthine - guanine - phosphoribosyltransferase (HGPRT) and 5'-nucleotidase activities. Nevertheless these enzymatic activities could be detected by the modification of culture conditions or treatment of crude extracts from Drosophila cells (1). A Drosophila melanogaster hemocyte line enabled demonstration of a relationship between the activities of HGPRT and 5'-nucleotidase. Only the addition of azaserine to the culture medium allows detection of a measurable amount of HGPRT and 5'-nucleotidase in cells devoid of endogenous synthesis of purine nucleotides. This action of azaserine suggests that the simultaneous expression of both enzymes is first directed by the suppression of the 5'-nucleotidase inhibition by pyrimidine nucleotides, and later on by the 5'-nucleotidase activity hydrolyzing the mononucleotides which inhibited the HGPRT, resulting its expression. This regulation system is confirmed by the simultaneous recovery of HGPRT and 5'-nucleotidase activities after heating cell cultures or cell extracts. Under such experimental conditions, 5'-nucleotidase is desensitized towards its negative effectors, while HGPRT regulation remains unchanged.

Erythrocyte disorders of purine and pyrimidine metabolism.

The maturing reticulocyte degrades ribosomal RNA to constituent ribonucleoside phosphates. Guanosine ribonucleotides are retained only in small amounts and pyrimidine ribonucleotides only in trace quantities. In the mature erythrocyte more than 97% of total nucleotides are the interconvertible adenosine mono-, di-, and triphosphates. High energy ATP fuels most of the reactions required to sustain viability. Unable to synthesize adenosine phosphates from small precursor molecules, the red cell relies on certain salvage pathways to replenish its losses from the adenosine phosphate pool. The most important of these involve adenosine. Adenylate kinase deficiency, when severe, is associated with nonspherocytic hemolytic anemia. A genetically-determined deficiency of pyrimidine 5'-nucleotidase prevents the normal dephosphorylation of pyrimidine ribonucleotides, and hence is characterized by the unique accumulation of pyrimidine phosphates intracellularly. Other features are chronic hemolytic anemia, splenomegaly, and a profound increase in basophilic stippling on the stained blood film. The syndrome is transmitted as an autosomal recessive disorder. A similar syndrome is found in severe lead poisoning as a consequence of nucleotidase inhibition by lead. An inherited, dominantly transmitted hemolytic anemia associated with low red cell ATP and a 45-70 fold increase in the enzymatic activity of adenosine deaminase has also been documented. The undefined molecular lesion appears to involve overproduction of an entirely normal enzyme protein. Severe deficiency of either of two sequential enzymes of purine metabolism, adenosine deaminase anemia, but by excessive accumulations of deoxyribonucleotides within red cells and lymphocytes. The clinical counterpart of each is a severe immunodeficiency state secondary to lymphopenia and lymphocyte dysfunction. Certain other rare clinical syndromes involving disturbed nucleotide metabolism also are detectable by red cell assay procedures.

Effects of Con A and other lectins on pure 5′nucleotidase isolated from lymphocyte plasma membranes

Inhibition of purified or membrane-bound 5′nucleotidase by various lectins was studied in lymphocytes from pig mesenteric lymph nodes. Con A or Lens culinaris lectin LcH inhibited (75 %) purified 5′nucleotidase by a non-competitive process without cooperativity. Inhibition by these lectins of 5′ nucleotidase activity in whole lymphocytes, plasma membranes (untreated or solubilized) and LcH-receptor fraction displayed high positive cooperativity, reached higher level (90 %) and was of mixed type. An interaction between lectin receptors and 5′nucleotidase accounted for these differences. Wheat germ agglutinin (WGA) and divalent Con A which are not mitogenic for T lymphocytes had no effect on 5′nucleotidase; pokeweed mitogen (PWM), mitogen of T and B cells, was not inhibitor. When membrane proteins were cross-linked by glutaraldehyde, Con A inhibition of whole lymphocyte 5′nucleotidase presented the same properties as the purified enzyme. Possible correlation between 5′nucleotidase inhibition and lymphocyte stimulation is discussed.