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PubMed1993

Purification and regulation of an AMP-specific cytosolic 5'-nucleotidase from dog heart.

The major enzyme responsible for adenosine production during myocardial hypoxia or ischemia is 5'-nucleotidase. We purified an AMP-specific 5'-nucleotidase to homogeneity from the 150,000-g supernatant of dog heart homogenate using phosphocellulose, DEAE-cellulose, and ADP-agarose affinity chromatography. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of the purified enzyme yielded a single protein band of 43 kDa. The molecular mass of the holoenzyme, determined by gel filtration and sucrose density-gradient centrifugation, was approximately 166 kDa, suggesting a tetrameric structure. Dog heart cytosolic 5'-nucleotidase was active at physiological pH (6.8-7.8) and demonstrated a preference for AMP over IMP as substrate. The enzyme exhibited sigmoidal saturation kinetics, with half-maximal activity at 2.6 mM AMP in the absence of ADP. ADP (0-250 microM) activated cytosolic 5'-nucleotidase by increasing maximal velocity and affinity for AMP. The enzyme was inhibited by 4 mM ATP, but 5'-nucleotidase activity increased as [ATP] was reduced. Mg2+ was required for activity, with maximal activation at approximately 3.5 mM free Mg2+. These data suggest that the regulation of AMP-specific cytosolic 5'-nucleotidase by adenine nucleotides and free Mg2+ may be important in the production of adenosine during conditions promoting ATP hydrolysis, such as myocardial hypoxia or ischemia.

5'-Nucleotidase↗

PubMed1996

Cardioprotection due to preconditioning correlates with increased ecto-5'-nucleotidase activity.

We investigated whether loss of myocardial protection after ischemic preconditioning (IP) is related to the extent of deactivation of activated ecto-5'-nucleotidase. The coronary arteries of mongrel dogs were occluded four times for 5 min separated by 5 min of reperfusion (IP). Five (IP1), 30 (IP2), 60 (IP3), and 120 min (IP4) after the fourth 5-min coronary occlusion or after a corresponding nonischemic period (control groups), the coronary arteries were occluded for 90 min followed by 6 h of reperfusion. The infarct size-limited effect of IP gradually disappeared in the IP2 (21.6 +/- 3.9%) and IP3 (33.8 +/- 3.6%) groups compared with the IP1 group (8.3 +/- 1.6%) and returned to the control level in the IP4 group (39.9 +/- 5.2%). The increased ecto-5' -nucleotidase activity due to the IP procedure decreased according to the order of IP1 to IP4 groups. Infarct size was inversely correlated with ecto-5'-nucleotidase activity (P < 0.001). An inhibitor of ecto-5'-nucleotidase blunted the infarct size-limiting effect of IP. The infarct size-limiting effect of IP decreased as the activation of ecto-5'-nucleotidase was blunted. These results suggest that ecto-5'-nucleotidase activity plays a key role in the cardioprotection of IP.

5'-Nucleotidase↗

PubMed2002

Plasma 5'-nucleotidase activities and uric acid levels in women with pre-eclampsia.

The present study investigated plasma activity of 5'-nucleotidase, a key enzyme in the production of adenosine, in pre-eclampsia, and evaluated the relationship between changes in 5'-nucleotidase activity, and levels of uric acid, endproduct of the purine metabolism, and the severity of pre-eclampsia. We measured plasma 5'-nucleotidase activities and uric acid levels in women with 18 normal pregnancies, mild and severe pre-eclampsia. In mild and severe pre-eclampsia, plasma 5'-nucleotidase activities and uric acid levels were significantly increased compared with those in normal pregnancy (p < 0.05). Plasma 5'-nucleotidase activity increased according to increases in uric acid levels and the severity of pre-eclampsia. These results suggest that increased plasma 5'-nucleotidase activity may, at least in part, be related to changes in purine metabolism in pre-eclampsia.

5'-Nucleotidase↗

PubMedDate not supplied

Anemia induces 5'-nucleotidase in fibroblasts of cortical labyrinth of rat kidney.

We recently observed a strong increase of 5'-nucleotidase in renal fibroblasts of rats that were anemic due to an immunity against erythropoietin. In order to test if the change of 5'-nucleotidase was related with anemia, we studied the distribution of the enzyme in irradiated rats treated with phenylhydrazine. The hematocrit of these rats decreased to 15% within 4 days and erythropoietin levels were more than 200 times over controls. After 7 days a histochemical study showed that the enzymatic activity and the immunoreactivity for 5'-nucleotidase was markedly enhanced in the fibroblasts of the cortical labyrinth. There was no modification of 5'-nucleotidase in other cell types of the kidney. The 5'-nucleotidase activity of renal fibroblasts in cell culture increased by 72% upon addition of 160 microM 5'-AMP to the culture medium for 8 days. We propose that anemia provokes an energy deficit in some structure in the cortical labyrinth. This might increase the concentration of 5'-AMP which would induce 5'-nucleotidase. An interesting consequence of these events would be an increased production of adenosine in the direct vicinity of some of its putative targets, the glomerular arterioles and the erythropoietin-producing cells.

5'-Nucleotidase↗

PubMed1992

Superoxide dismutase enhances ischemia-induced reactive hyperemic flow and adenosine release in dogs. A role of 5'-nucleotidase activity.

To test the hypothesis that 5'-nucleotidase activity during ischemia is attenuated by oxygen-derived free radicals, we measured ischemia-induced reactive hyperemic flow, adenosine release, and 5'-nucleotidase activity in dogs (n = 62). A 1-minute occlusion of the coronary artery caused reactive hyperemic flow (307 +/- 5 versus 92 +/- 1 ml.100 g-1.min-1 at baseline) with increased release of adenosine (14.4 +/- 1.4 versus 0.4 +/- 0.1 nmol.100 g-1.min-1 at baseline). Superoxide dismutase augmented (p less than 0.001) both peak coronary blood flow (333 +/- 6 ml.100 g-1.min-1) and repayment (436 +/- 12 versus 320 +/- 7 ml/100 g in the untreated group). Adenosine release during reperfusion was augmented (22.7 +/- 1.9 nmol.100 g-1.min-1, p less than 0.001), and 8-phenyltheophylline completely abolished the enhanced reactive hyperemia. Enzymatic assay of 5'-nucleotidase activity revealed that the administration of superoxide dismutase increases ecto-5'-nucleotidase activity in ischemic myocardium. When an inhibitor of ecto-5'-nucleotidase, alpha, beta-methyleneadenosine 5'-diphosphate, was administered, the effects of superoxide dismutase were completely abolished. Thus, we conclude that 1) the augmentation of reactive hyperemic flow caused by superoxide dismutase is attributed to the enhanced release of adenosine and 2) the enhanced release of adenosine over the untreated controls is attributed to the protection of ecto-5'-nucleotidase activity during ischemia.

5'-Nucleotidase↗

PubMed2003

The yeast ISN1 (YOR155c) gene encodes a new type of IMP-specific 5'-nucleotidase.

BACKGROUND: The purine salvage enzyme inosine 5'-monophosphate (IMP)-specific 5'-nucleotidase catalyzes degradation of IMP to inosine. Although this enzymatic activity has been purified and characterized in Saccharomyces cerevisiae, the gene encoding IMP 5'-nucleotidase had not been identified. RESULTS: Mass spectrometry analysis of several peptides of this enzyme purified from yeast allowed identification of the corresponding gene as YOR155c, an open reading frame of unknown function, renamed ISN1. The deduced Isn1p sequence was clearly not homologous to 5'-nucleotidases from other species. However, significant similarities to Isn1p were found in proteins of unknown function from Neurospora crassa, Plasmodium falciparum and several yeast species. Knock-out of ISN1 resulted in the total loss of IMP-specific 5'-nucleotidase activity, thus confirming that the ISN1 gene indeed encodes the enzymatic activity purified from yeast. In vivo studies revealed that, when IMP is overproduced through constitutive activation of the IMP de novo synthesis pathway, ISN1 is required for excretion of inosine and hypoxanthine in the medium. CONCLUSION: We have identified a new yeast gene, ISN1 (YOR155c), as encoding IMP-specific 5'-nucleotidase activity. The ISN1 gene defines a new type of 5'-nucleotidase which was demonstrated to be functional in vivo.

5'-Nucleotidase↗

PubMed1976

A comparative study of histochemical mapping on the distribution of acid phosphatase and 5-nucleotidase in the forebrain of frog (Rana tigrina).

The four well defined dorsal, lateral, medial and intermediate olfactory tracts of the frog have been studied. During their courses they become intermingled with fibers related to the primordial septum, the primordial hippocampus, the primordial dorsal pallium and the primordial piriform areas. In acid phosphatase preparations all these tracts are completely negative, and, therefore, are not identifiable. However, these tracts have intense staining reaction to 5-nucleotidase throughout their courses. Differences in the distribution of acid phosphatase and 5-nucleotidase are apparent in the hippocampal and anterior commissures. These are negative for acid phosphatase. In 5-nucleotidase preparations the anterior commissure has an intensely positive whereas the hippocampal commissure has a completely negative reaction. The medial septal nucleus is intensely positive for 5-nucleotidase at the anterior hemisphere level. There is a gradual decrease in intensity of staining in this nucleus towards the posterior hemisphere levels so that in the last few sections of the forebrain of the frog the medial septal nucleus is completely unstained or negative. In the acid phosphatase preparations at all levels the medial septal nucleus presents a moderately positive reaction. In 5-nucleotidase preparations, the dorsomedial part of primordial hippocampus is intensely positive whereas the dorsal part of the hippocampus is completely negative. In acid phosphatase preparations, both dorsal and dorsomedial parts of hippocampus are intensely positive. The striatal complex and the lateral septal nucleus are positive for acid phosphatase and negative for 5-nucleotidase. The possible significance of some of these similarities and differences in tracts and nuclei has been considered with reference to the functioning of the various parts. The present histoenzymological preparations have revealed distinct nerve pathways. In some instances clear-cut connections, which had hitherto been unrevealed by neuroanotomical methods, have been demonstrated.

Acid Phosphatase↗

PubMed1975

[Light and electron microscopic localization of enzymes: 5'-nucleotidase (author's transl)].

5'-nucleotidase (EC 3.1.3.5), an important enzyme in the metabolism of nucleotides, is generally accepted as a plasma membrane marker. The enzyme selectively splits phosphoric acid from 5' mononucleotides. Several methods are available for the histochemical localization of enzymes (antigenic properties of the enzyme protein, enzyme properties and activity and labelled specific inhibitors). Only the method based on enzyme properties has been used up to now in the case of 5'-nucleotidase. Free phosphoric acid liberated during the dephosphorylation of substrates such as AMP or IMP is rendered visible at the sites of 5' nucleotidase activity in the tissue by precipitation as lead or calcium phosphate. An improvement in the light microscopic technique is achieved by the use of freezedried tissue embedded in glycol methacrylate, whereby the histochemical reaction can be performed on semi-thin sections. Since lead phosphate is electron dense, these precipitates can easily be detected in the electron microscope too. Wide species and organ differences are found with respect to the distribution of 5'-nucleotidase activity. The well-known localization of the enzyme on the outer cell surface according to biochemical studies is confirmed by electron microscopic findings. A purely catabolic function of 5'-nucleotidase, as propounded in the literature, seems dubious since high 5'-nucleotidase activity was demonstrated in rapidly proliferating tissue too.

Animals↗

PubMed1992

Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum.

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.

5'-Nucleotidase↗

PubMed1992

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.

5'-Nucleotidase↗

PubMed1978

Further studies on 5'-Nucleotidase from serum of liver cirrhotic individuals.

The kinetic properties of 5'-Nucleotidase were investigated in untreated patients with liver cirrhosis at 37 degrees C. Mg+2 and Mn+2 were found to activate both normal and liver cirrhotic 5'-Nucleotidase, but Nickel inhibited the enzyme in both systems competitively. Both ATP and adenosine act as inhibitors to 5'-Nucleotidase. The inhibitory constant for ATP was different in normal and liver cirrhotic individuals, 0.1 +/- 0.03 for normal and 0.225 +/- 0.02 for liver cirrhosis. In our investigation, ATP was found to be a competitive inhibitor of 5'-Nucleotidase which compete the substrate (A-5'-MP) for the active site. Inhibition of 5'-Nucleotidase by adenosine is of non-competitive type, for both normal and liver cirrhotic sera. It was observed that both serum 5'-Nucleotidase exhibited pH dependent characteristics; in that there was an optimum substrate concentration at each pH value and the plot of pKm versus pH shows great dependency of km on pH.

Adenosine↗

PubMed1977

Inhibition by concanavalin A as the basis for a specific assay of serum 5'-nucleotidase activity.

Concanavalin A inhibits serum 5'-nucleotidase activity, without causing significant inhibition of alkaline phosphatase activity. This observation serves as the basis for a new method for assaying the 5'-nucleotidase activity in serum, which depends upon the difference between the enzymic hydrolysis of adenosine-5'-monophosphate in the presence and absence of concanavalin A. A denosine released by the 5'-nucleotidase reaction is deaminated by a coupled reaction with adenosine deaminase to liberate inosine and ammonia, and ammonia is measured colorimetrically by the Berthelot reaction. In sera from 40 healthy adult persons, 5'-nucleotidase activity averaged 6.4 U/liter (SD, +/-2.0; range, 3-12). In sera from 100 patients, measurements of 5'-nucleotidase activity by the new assay averaged 8% lower than by a generally accepted method in which phenyl phosphate is used to suppress hydrolysis of adenosine-5'-monophosphate by alkaline phosphatase activity. The clinical validy of the new assay was tested by measuring serum 5'-nucleotidase activities in rats with bile duct ligation and in rats treated with thioacetamide to induce hepatocellular injury.

Adult↗

PubMed1997

Effects of triton X-100 and concanavalin A on the properties of 5'-nucleotidase in rat liver and adipose plasma membranes: a role of membrane structure in the regulation of enzyme activity.

The kinetic and thermodynamic properties of 5'-nucleotidase (EC 3.1,3.5) were investigated in rat liver and adipose plasma membranes (PM) after their structural modification with nonionic detergent Triton X-100 (TX-100) or lectin concanavalin A (Con-A). The apparent K(m) value of 5'-nucleotidase decreased after PM treatment with subsolubilizing TX-100 concentrations (0.005-0.015%) and then tended to grow at higher TX-100 concentrations (0.03-0.05%). Treatment of PM with Con-A resulted in the noncompetitive inhibition of 5'-nucleotidase in a dose-dependent manner. The Arrhenius plot of the 5'-nucleotidase activity in the control PM exhibited a single well defined break at about 28-31 degrees C with a lower activation energy at the upper slope of the graph. The shape of the Arrhenius graphs and the thermodynamic parameters of 5'-nucleotidase were specifically modified upon the PM treatment with increasing concentrations of TX-100 or Con-A. It was suggested that the functional activity of 5'-nucleotidase and its conformation within the membrane matrix may be directly regulated by the structural states of the enzyme specific microenvironment and of the membrane lipid bilayer. Moreover, the existence of the spatial and/or compositional optima for the relationship between the enzyme molecule and its lipid surrounding may be reasonably supposed.

5'-Nucleotidase↗

PubMed1997

Role of protein kinase C-alpha in activation of ecto-5'-nucleotidase in the preconditioned canine myocardium.

We have reported that activation of protein kinase C (PKC) increases ecto-5'-nucleotidase activity, which may contribute to the infarct size-limiting effect of ischemic preconditioning. Since we have reported that Ca(2+)- and phospholipid-sensitive PKC is activated due to ischemic preconditioning, we further tested 1) whether PKC-alpha or -beta is translocated to the cellular membrane of the preconditioned canine myocardium, and 2) whether activation of PKC contributes to the increase in ecto-5'-nucleotidase activity via phosphorylation-dependent mechanisms. Four times of 5 minutes coronary occlusion separated by 5 minutes of reperfusion (ischemic preconditioning) translocated PKC-alpha to the cellular membrane in the canine hearts, although PKC-beta, -delta, -epsilon, and -zeta were not translocated. The activity of Ca(2+)- and phospholipid-sensitive PKC increased, which was attenuated by the removal of either Ca2+ or phosphatidylserine. Ecto-5'-nucleotidase was also activated in the preconditioned myocardium compared with control. Inhibition of PKC due to GF109203X blunted the activation of myocardial ecto-5'-nucleotidase. Okadaic acid (an inhibitor of phosphatase) enhanced the increases in ecto-5'-nucleotidase activity due to preconditioning, and this enhancement was blunted by GF109203X. We conclude that ischemic preconditioning activates PKC-alpha, and thus ecto-5'-nucleotidase.

Adenosine Triphosphatases↗

PubMed2000

Ecto-5'-nucleotidase plays a role in the cardioprotective effects of heat shock protein 72 in ischemia-reperfusion injury in rat hearts.

OBJECTIVE: Heat shock protein 72 (HSP72) is involved in the myocardial self-preservation system under several conditions such as ischemia-reperfusion injury or late preconditioning. However, its mechanism is not fully understood. Ecto-5'-nucleotidase is a key enzyme for synthesizing adenosine and plays an important role in ischemic preconditioning. In this study, we tested the hypothesis that ecto-5'-nucleotidase plays a role in the cardioprotection of HSP72. METHODS: Rat hearts (H group, n=6) were transfected with HSP72 gene by an intracoronary infusion of hemagglutinating virus of Japan (HVJ)-liposome complex. Control hearts (C group, n=6) were transfected with the beta-galactosidase gene. Following 30 min of normothermic ischemia, grafts were reperfused using Langendorff apparatus. RESULTS: The activity of ecto-5'-nucleotidase was significantly higher in H group than C group both before and after ischemia-reperfusion (H vs. C; 0.51+/-0.05 vs. 0.29+/-0.06, and 1.41+/-0.15 vs. 0.85+/-0.11 nmol/mg protein/min, P<0.05). H group also showed significant better functional recoveries than C group (P<0.05), as well as less creatine phosphokinase leakage (4.4+/-2.8 vs. 14.2+/-3.4 mU/min, P<0.05) and higher adenosine release (247.5+/-35.1 vs. 54.3+/-1.7 pmol/min, P<0.05). Administration of alpha,beta-methylene adenosine diphosphate (AMP-CP), an inhibitor of ecto-5'-nucleotidase, significantly diminished the tolerance to ischemia-reperfusion injury in H group (P<0.05). CONCLUSION: These results demonstrated that ecto-5'-nucleotidase activated by an overexpression of HSP72 attenuated ischemia-reperfusion injury in the rat myocardium. They suggest that ecto-5'-nucleotidase plays a role in the cardioprotective effects of HSP72 in rat hearts.

Adenosine Diphosphate↗

PubMed2004

Site-directed mutagenesis of human soluble calcium-activated nucleotidase 1 (hSCAN-1): identification of residues essential for enzyme activity and the Ca(2+)-induced conformational change.

Human soluble calcium-activated nucleotidase 1 (hSCAN-1) is the human homologue of soluble apyrases found in blood-sucking insects. This family of nucleotidases is unrelated in sequence to more well-studied nucleotidases, and very little is known about the enzymatic mechanism. By multiple sequence alignment, eight regions that are highly conserved in the hSCAN-1 family were identified and named. To identify amino acids important for catalytic activity and enzyme specificity, seven point mutations were constructed, expressed in bacteria, refolded, purified, and characterized. Substitution of glutamic acid 130 with tyrosine resulted in dramatically increased nucleotidase activities, while mutagenesis of aspartic acid 151 to alanine and aspartic acid 84 to alanine completely abolished activity. Mutagenesis of arginine 133 and arginine 271 resulted in enzymes with very little nucleotidase activity. Mutagenesis of aspartic acid 175 to alanine and glycine 122 to glutamic acid had smaller negative effects on enzyme activities. Previously, our laboratory showed that calcium triggers a conformational change in hSCAN-1 necessary for nucleotidase activity. Here we show that several mutants (D84A, R133A, and D151A) that lost most of their activity were unable to undergo the conformational change induced by Ca(2+), as shown by Cibacron blue binding, limited proteolysis, and tryptophan fluorescence. We conclude that aspartic acid residues 84 and 151, as well as arginine residue 133, are essential for the Ca(2+)-induced conformational change that is necessary for enzyme activity. Aspartic acid 175 and glutamic acid 130 are important for determining substrate specificity. In addition, we show that Sr(2+), unlike Mg(2+) and other divalent cations, can substitute for Ca(2+) to induce the conformational change necessary for enzyme activity. However, Sr(2+) cannot substitute for Ca(2+) to support nucleotide hydrolysis, presumably because Sr(2+) cannot substitute for Ca(2+) in its second role as a nucleotide cosubstrate. The ramifications of our results on the interpretation of a recently published crystal structure are discussed. This information will facilitate future engineering of this enzyme designed to enhance its ability to hydrolyze ADP and thus increase its potential for therapeutic use in the treatment of pathological ischemic events triggered via activation of platelets by ADP.

Amino Acid Sequence↗

PubMed1985

Subpopulations of fibroblasts from mouse skeletal muscle defined by clonal variation for 5' nucleotidase expression.

A primary cloning technique has been employed for the isolation of nine spontaneously transformed cell lines from mouse skeletal muscle. Four of these lines were isolated after selection for partial resistance to the purine (adenine) analog 2'6'diaminopurine and five were isolated from non-selected control dishes. Four of the nonselected lines and three of the selected lines demonstrated a fibroblastoid morphology in vitro. The other two cell lines (one from each group) were epithelioid. Two of the three selected fibroblastoid lines were found to contain significant quantities of the enzyme 5'nucleotidase (EC3.1.3.5), whereas the four nonselected fibroblastlike lines, one selected fibroblastlike line, and the two epithelioid lines did not. In the two cell lines expressing 5'nucleotidase activity, this expression was stable in the absence of selective pressure. Histochemical staining of mouse skeletal muscle for 5'nucleotidase activity demonstrated positive staining in the cells of small blood vessels and in a subset of the connective tissue cells. The bulk of the skeletal muscle tissue, however, had no detectable 5'nucleotidase activity. We propose that the two cultivatable types of fibroblastoid cell lines represent distinct classes of fibroblastlike cells in vivo, reflecting alternative states of stable cellular differentiation involving 5'nucleotidase expression.

2-Aminopurine↗

PubMed1982

Localization of adenylate cyclase and 5'-nucleotidase activities in human thyroid follicular cells.

The localization of adenylate cyclase and 5'-nucleotidase activities in the follicular cells of adenomatous goiter and normal thyroid was studied by light and electron microscopy. Simultaneous biochemical measurement for both activities was carried out to confirm the histochemical findings. Adenylyl-imidodiphosphate (AMP-PNP) was used as an effective substrate for adenylate cyclase. The specificity of the adenylate cyclase reaction was also examined by adding oxalacetic acid or PCMB as an adenylate cyclase inhibitor, and by adding sodium fluoride or TSH as an adenylate cyclase stimulator to the reaction mixture. In the case of tissue from adenomatous goiter, a large amount of the reaction product of the adenylate cyclase activity was found uniformly in the apical and lateral plasma membrane and not in the basal plasma membrane. In the cases of normal thyroid, a small amount of the reaction product of adenylate cyclase activity was demonstrated, and only in the lateral plasma membrane of the follicular cells. On the otherhand, the histochemical localization of 5'-nucleotidase activity was the same in adenomatous goiter and normal thyroid. The reaction product of 5'-nucleotidase activity was found predominantly in the apical plasma membrane of the follicular cells. The biochemical findings indicated that the activity of adenylate cyclase per gram tissue was approximately 2 times higher in the case of adenomatous goiter than that in the case of normal thyroid, while the 5'-nucleotidase activity in adenomatous goiter was in slightly higher level than in normal thyroid. Thus the histochemically demonstrable amount of adenylate cyclase and 5'-nucleotidase reflected the activity levels measured biochemically. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that this structure may not play any important role in TSH reception.

5'-Nucleotidase↗