Absence of 5′-nucleotidase in porcine polymorphonuclear leucocyte membranes

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess $\text{p-}\text{nitrophenyl}$ phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5′-nucleotidase inhibitor α,β-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5′-nucleotidase. An antibody against mouse liver 5′-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5′-nucleotidase and the necessity for distinguishing between true 5′-nucleotidase and non-specific phosphatase activity is discussed.     

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.     

Ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio)

Adenosine, a well-known neuromodulator, may be formed intracellularly in the CNS from degradation of AMP and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. This study reports the enzymatic properties of an ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for AMP hydrolysis in a pH range of 7.0-7.5 in the presence of Mg(2+). The enzyme presented a maximal activity for AMP hydrolysis at 37 degrees C. The apparent K(m) and V(max) values for Mg(2+)-AMP were 135.3+/-16 microM and 29+/-4.2 nmol Pi.min(-1).mg(-1) protein, respectively. The enzyme was able to hydrolyze both purine and pyrimidine monophosphate nucleotides, such as UMP, GMP and CMP. Levamisole and tetramisole (1 mM), specific inhibitors of alkaline phosphatases, did not alter the enzymatic activity. However, a significant inhibition of AMP hydrolysis (42%) was observed in the presence of 100 microM alpha,beta-methylene-ADP, a known inhibitor of ecto-5'-nucleotidase. Since 5'-nucleotidase represents the major enzyme responsible for the formation of extracellular adenosine, the enzymatic characterization is important to understand its role in purinergic systems and the involvement of adenosine in the regulation of neurotransmitter release.     

Inhibition of 5′-nucleotidase from Ehrlich ascites-tumour cells by nucleoside triphosphates

1. 5'-Nucleotidase activity was obtained in a soluble form after treatment of a particulate fraction from Ehrlich ascites-tumour cells with deoxycholate. The relative rates of hydrolysis of 6-thioinosine 5'-phosphate, UMP, AMP, CMP, GMP, IMP, xanthosine monophosphate, thymidine monophosphate and 2',3'-AMP were 180, 129, 100, 93, 83, 79, 46, 41 and 3 respectively. 2. Values found for the Michaelis constant were: AMP, 67+/-12mum; IMP, 111+/-8mum; GMP, 93mum. 3. ATP and thymidine triphosphate were competitive inhibitors of AMP hydrolysis (inhibitor constants 0.4 and 4.8mum respectively); UTP, GTP and CTP were mixed competitive and non-competitive inhibitors. Thymidine triphosphate was a competitive inhibitor of IMP hydrolysis (inhibitor constant 14.4mum) and ATP, UTP and GTP showed mixed competitive and non-competitive inhibition. 4. ATP, thymidine triphosphate, UTP, GTP and CTP did not completely inhibit hydrolysis of AMP, IMP and UMP; the concentrations of ATP required to inhibit AMP and IMP hydrolysis by 50% were 12 and 230mum respectively. 5. Non-hyperbolic curves relating activity to UMP concentration were obtained in the presence and absence of triphosphates. 6. After fractionation on Sephadex G-200 columns a single peak of 5'-nucleotidase activity (particle weight 120000-125000) was obtained with AMP, IMP and GMP as substrates. UMP hydrolysis was catalysed by enzyme in this peak and in two slower peaks corresponding to apparent particle weights of 32000 and 16000; a single component (particle weight 120000), reacting with UMP and insensitive to UTP inhibition, was obtained when the column was eluted with buffer containing 1mm-UMP. 7. The possible significance of the results in the regulation of tumour-cell 5'-nucleotidase is discussed.     

Properties of 5'-nucleotidase in rat heart sarcolemma

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.     

A specific inhibitor of heart cytosolic 5'-nucleotidase I attenuates hydrolysis of adenosine 5'-monophosphate in primary rat myocytes

ATP breakdown was triggered in primary rat myocytes in the presence of coformycin to force the catabolism of AMP through hydrolysis to adenosine. Selective inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium were used to measure the intracellular contribution of this enzyme to AMP hydrolysis under these conditions. The selective inhibitor 5-ethynyl-2',3'-dideoxyuridine inhibited the hydrolysis of AMP to adenosine in a concentration-dependent manner with an IC50 value of 20 microM. Maximal inhibition prevented 76% of the conversion of AMP to adenosine, indicating that under these conditions the majority of AMP hydrolysis in rat myocytes occurs through this enzyme. When ATP breakdown was triggered in the presence of thymidine 5'-phosphonate, a more potent inhibitor of the purified cytosolic 5'-nucleotidase, less inhibition of AMP hydrolysis occurred and only after prolonged preincubation of the myocytes with the inhibitor. These data demonstrate that the selective nucleoside inhibitors of c-N-I can effectively block the hydrolysis of AMP inside myocytes. Thus, these inhibitors may be useful tools in identifying the role of c-N-I during ATP catabolism in whole tissue and animal experiments.     

Interaction of [3H]AMP with liver cells and their plasma membranes

Specific binding of [3H]AMP to rat hepatocytes and their plasma membranes was studied. It was shown that the time course of this binding reached a maximum within the first 15 seconds. An equilibrium binding study revealed the presence of a single class of binding sites with Kd of 20 microM both in hepatocytes and in plasma membranes. The [3H]AMP binding sites were inactivated by treatment with trypsin as well as by heating. 5'-Phosphorylated derivatives of adenosine (ATP, ADP) effectively competed with [3H]AMP for the binding sites, while adenosine, beta-glycerophosphate and 3'-AMP were inactive. The binding of [3H]AMP increased by 400% in the presence of concanavalin A, a specific inhibitor of plasma membrane 5'-nucleotidase. It was concluded that the catalytic center of 5'-nucleotidase is a receptor for adenine nucleotides.     

Ethanol and acetaldehyde alter NTPDase and 5'-nucleotidase from zebrafish brain membranes

Alcohol abuse is an acute health problem throughout the world and alcohol consumption is linked to the occurrence of several pathological conditions. Here we tested the acute effects of ethanol on NTPDases (nucleoside triphosphate diphosphohydrolases) and 5'-nucleotidase in zebrafish (Danio rerio) brain membranes. The results have shown a decrease on ATP (36.3 and 18.4%) and ADP (30 and 20%) hydrolysis after 0.5 and 1% (v/v) ethanol exposure during 60 min, respectively. In contrast, no changes on 5'-nucleotidase activity were observed in zebrafish brain membranes. Ethanol in vitro did not alter ATP and ADP hydrolysis, but AMP hydrolysis was inhibited at 0.5, and 1% (23 and 28%, respectively). Acetaldehyde in vitro, in the range 0.5-1%, inhibited ATP (40-85%) and ADP (28-65%) hydrolysis, whereas AMP hydrolysis was reduced (52, 58 and 64%) at 0.25, 0.5 and 1%, respectively. Acetate in vitro did not alter these enzyme activities. Semi-quantitative expression analysis of NTPDase and 5'-nucleotidase were performed. Ethanol treatment reduced NTPDase1 and three isoforms of NTPDase2 mRNA levels. These findings demonstrate that acute ethanol intoxication may influence the enzyme pathway involved in the degradation of ATP to adenosine, which could affect the responses mediated by adenine nucleotides and nucleosides in zebrafish central nervous system.     

A kinetic study of the soluble 5''-nucleotidase of rat liver.

1. The kinetic properties of the 5'-nucleotidase (EC 3.1.3.5) present in the cytosol of rat liver were investigated in relation to the conversion of adenine nucleotides into uric acid, with particular reference to the stimulation of this process by fructose. The enzyme was assayed by the release of Pi and by a new and more sensitive radiochemical procedure. 2. When IMP was used as substrate, the partially purified enzyme displayed almost hyperbolic kinetics (h = 1.1) with S0.5 = 1.2 mM. Similar kinetics were observed with GMP and other nucleoside 5'-monophosphates, except AMP. 3. Vmax. of the enzyme for AMP was about the same as for IMP, but the kinetics were sigmoidal (h = 1.6) with S 0.5 = 10 mM. 4. The hydrolysis of IMP was inhibited competitively by GMP. IMP, at concentrations up to 0.5 mM, had a paradoxical stimulatory action on the hydrolysis of 2-5 mM-AMP and was inhibitory at higher concentrations. 5. The activity of the enzyme towards AMP and IMP was stimulated by ATP and GTP, and inhibited by Pi. Activators and inhibitor approximately cancelled each others' effects. At pH 7.4, the enzymic activity with 0.2 mM-AMP was undetectable under physiological conditions. 6. It is concluded that, in the liver cell, AMP is not hydrolysed by the soluble 5'-nucleotidase, but that its degradation requires prior deamination to IMP.     

Cyclic AMP metabolism by swine adipocyte microsomal and plasma membranes

Extracellular cyclic AMP is source of extracellular adenosine in brain and kidney. Whether this occurs in adipose tissue is unknown. The present study evaluated the capacity of swine adipocyte plasma membranes to metabolize cyclic AMP to AMP and adenosine, via phosphodiesterase (PDE) and 5'-nucleotidase (5'-NT), respectively. Plasma membranes (PM) and microsomal membranes (MM) were isolated from over-the-shoulder subcutaneous adipose tissue of 3 month-old male miniature swine. The purity of the membrane fractions was determined and PDE and 5'-NT activities in PM and MM fractions were corrected for cross-contamination. The maximal activity of MM-PDE was 7-fold greater than that of PM-PDE. MM-PDE was 100% inhibited by 5 microM cilostamide, while PM-PDE was unaffected by this PDE3B inhibitor. Inhibitors of PDE1, PDE2, PDE4 and PDE5 also failed to inhibit PM-PDE. However, 1 mM DPSPX inhibited PM-PDE activity by 72%. When PM were incubated with 0.8 microM cyclic AMP for 20 min, AMP accumulation was four times that of adenosine. These data demonstrate that cyclic AMP can be converted to AMP and adenosine by the PM-bound enzymes 5'-NT and PDE, and suggest that the PM-PDE responsible for extracellular cyclic AMP metabolism to AMP is distinct from the intracellular MM-PDE.     

5'-Nucleotidase activity of isolated mature rat cardiac myocytes

Specific location of 5'-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5'-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5'-nucleotidase is accessible to extracellular AMP. Inhibitors of 5'-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5'-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.     

Redox-dependent activation of 5'-nucleotidase in rat liver plasma membranes

Addition of NADH, but not NAD+ or NADPH, to rat liver plasma membranes resulted in the increase of their 5'-nucleotidase activity. NADH-dependent activation of 5'-nucleotidase was significantly suppressed by atebrine, an inhibitor of NADH dehydrogenase of plasma membranes, and completely abolished by 2,4-dinitrophenol (2 X 10(-4)M) and Triton X-100 (2%). Inhibitors of electron transfer in the mitochondrial respiratory chain, rotenone and potassium cyanide, failed to affect 5'-nucleotidase activity in both the presence and absence of NADH. The data obtained give reasons to suggest a redox-dependent mechanism of 5'-nucleotidase activation in rat liver plasma membranes.     

Biochemical properties of Candida parapsilosis ecto-5'-nucleotidase and the possible role of adenosine in macrophage interaction

Candida parapsilosis is considered to be an emerging fungal pathogen because it is associated with an increasing range of infections. In this work, we biochemically characterized ecto-5'-nucleotidase activity on the surface of living, intact C. parapsilosis cells. At a pH of 4.5, intact cells were able to hydrolyze 5'-AMP at a rate of 52.44 ± 7.01 nmol Pi h(-1) 10(-7) cells. 5'-AMP, 5'-IMP and 5'-UMP were hydrolyzed at similar rates, whereas 5'-GMP and 5'-CMP hydrolyzed at lower rates. Enzyme activity was increased by about 42% with addition of Mg(2+) or Ca(2+), and the optimum pH was in the acidic range. An inhibitor of phosphatase activities, sodium orthovanadate, showed no effect on AMP hydrolysis; however, as expected, ammonium molybdate, a classical nucleotidase inhibitor, inhibited the activity in a dose-dependent manner. The results indicated that the existence of an ecto-5'-nucleotidase could play a role in the control of extracellular nucleotide concentrations.     

Effect of sodium deoxycholate on 5'-nucleotidase.

The 5'-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5'-nucleotidase exhibited the same properties as the 5'-nucleotidase in plasma membranes. The 5'-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and Km. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p<0.01). There were no significant differences between different groups in the activities of cN-I, whole homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities of whole homogenate nucleotidase, total cN and cN-I. The results indicate no changes in the relative expression or catalytic behaviour of cN-I in AMP deaminase-deficient human skeletal muscle, but suggest that increased turnover of AMP by cN-I in working skeletal muscle is due to higher substrate availability of AMP.     

A Low-Km 5'-Nucleotidase from Rat Brain Cytosolic Fraction: Purification, Kinetic Properties, and Description of Regulation by a Novel Factor that Increases Sensitivity to Inhibition by ATP and ADP

Abstract: A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000- g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low K _m for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by α,β-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of M_r = 54,300 ± 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- K _m"soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000- g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.     

3-Phosphono-2-imino-4-oxoimidazoline--a competitive substrate of AMP-dephosphorylating enzymes from the cytosol fraction of the rat heart

3-Phosphono-2-imino-1-methyl-4-oxoimidazolidine (PIMOI), AMP and p-nitrophenyl phosphate (pNPP) were dephosphorylated in the presence of rat heart cytosol at 37 degrees C pH 6.3 at the rates of 0.71, 0.45 and 1.07 mumol/mg X h, respectively. When mixed together, these compounds inhibited the hydrolysis of each other, which points to the participation of common enzyme(s) in this process. The inhibitor of 5'-nucleotidase (alpha,beta-methylene)-ADP, did not affect PIMOI cleavage and moderately inhibited AMP hydrolysis (by ADP, did not affect PIMOI cleavage and moderately inhibited AMP hydrolysis (by 30-50%), thus suggesting that acidic phosphatases are responsible for PIMOI and AMP hydrolysis under these conditions (pH 6.3). Phosphocreatine (PCr) and phosphocyclocreatine (PcCr) were stable to hydrolysis by the cytosolic fraction. However, addition of AMP to the medium containing PCr or PcCr resulted in AMP phosphorylation down to ATP due to the effects of these phosphagens and, probably, of microcontaminations of ATP. This was followed by gradual disappearance of PCr or PcCr and by accumulation of Pi as a result of the "ATPase" activity in the cytosol. The hydrolysis of AMP, PIMOI and p-NPP was sensitive to sulfhydryl reagents [5,5'-dithio-bis-(2-nitrobenzoate) and, in part, 2,4-dinitro-fluorobenzene] and fluoride ion. Thus, PIMOI is a competitive substrate of acidic phosphatases in heart cytosol with respect to AMP and p-NPP. This may partly explain the protective effect of PIMOI on ischemic myocardium.     

5'-Nucleotidase activity and adenosine production in rat liver mitochondria.

The controversial subject of mitochondrial 5'-nucleotidase in the liver was studied employing density gradient fractionation combined with a method for analyzing the distribution profiles of marker enzymes based on multiple regression analysis. Triton WR-1339 was used to improve the separation of mitochondria from lysosomes by the gradient centrifugation technique. Adenosine production was examined further using acetate to increase intramitochondrial AMP, and thus adenosine production, in incubations with gradient centrifugation-purified mitochondria. Distribution analysis of the crude homogenate showed that 5'-nucleotidase activity exists in the mitochondrial fraction. To increase the resolution of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied. In this case the relative mitochondrial activity decreased but 5'-nucleotidase activity was still clearly detectable. The mitochondrial 5'-nucleotidase exhibited a Km of 94 microM and a Vmax of 31 nmol/min per mg protein for AMP. The kinetic data for the Mg2+, ATP, ADP and AOPCP sensitivity of the enzyme showed that it differs from the plasma membrane, lysosome and cytosol 5'-nucleotidases. AOPCP was only a moderate inhibitor, and ATP was a more potent inhibitor than ADP at a 1 mM concentration. The enzyme also showed a requirement of Mg2+. Acetate caused the conversion of intramitochondrial adenylates to AMP and the formation of adenosine. Adenosine concentration increased in the extramitochondrial space in a time-dependent manner, but only trace amounts of nucleotides were detected. The data show that 5'-nucleotidase activity producing adenosine exists in rat liver mitochondria and a concentration-dependent adenosine output from mitochondria by diffusion or facilitated diffusion is also suggested.     

In vitro exposure of heavy metals on nucleotidase and cholinesterase activities from the digestive gland of Helix aspersa

Zinc, copper and cadmium are important environmental contaminants and differences in purinergic and cholinergic systems of invertebrates have been described when compared to characteristics of these signaling systems in vertebrates. Here we evaluate the effect in vitro of these metals on the ATPase, 5'-nucleotidase and cholinesterase (ChE) activities in the digestive gland of Helix aspersa. Zinc (500 and 1000 microM) promoted a significant decrease in 5'-nucleotidase activity. However, it did not induce changes in ATP hydrolysis. Copper (25 and 50 microM), inhibited significantly ATPase activity, but did not alter 5'-nucleotidase when compared to control (no metal added). In relation to effects of cadmium, an inhibitory effect on ATP hydrolysis has been observed at concentrations of 100, 500 and 1000 microM and a similar decrease of AMP hydrolysis was observed at 500 and 1000 microM. However, there were no significant changes in ChE activity from homogenates of the digestive gland of H. aspersa for all metals tested. This study demonstrated that zinc, cadmium and copper affect ATPase and 5'-nucleotidase in digestive gland, but not ChE, suggesting that the purinergic system may be a target related to toxicity induced by these metals and a possible indicator of biological impact of exposure to these contaminants.     

Affinity labelling of 5''-nucleotidases with 5''-p-fluorosulphonylbenzoyladenosine.

5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membrane-bound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). 5'-FSBA irreversibly inactivates 5'-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5'-nucleotidase, abolished the inactivation by 5'-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5'-FSBA, which was employed for the radiolabelling of chicken gizzard 5'-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5'-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5'-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5'-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5'-nucleotidase by 5'-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.