Leishmania amazonensis impairs DC function by inhibiting CD40 expression via A2B adenosine receptor activation.

Dendritic cells (DCs) play an essential role in the modulation of immune responses and several studies have evaluated the interactions between Leishmania parasites and DCs. While extracellular ATP exhibits proinflammatory properties, adenosine is an important anti-inflammatory mediator. Here we investigated the effects of Leishmania infection on DC responses and the participation of purinergic signalling in this process. Bone marrow-derived dendritic cells (BMDCs) from C57BL/6J mice infected with Leishmania amazonensis, Leishmania braziliensis or Leishmania major metacyclic promastigotes showed decreased major histocompatibility complex (MHC) class II and CD86 expression and increased ectonucleotidase expression as compared with uninfected cells. In addition, L. amazonensis-infected DCs, which had lower CD40 expression, exhibited a decreased ability to induce T-cell proliferation. The presence of MRS1754, a highly selective A(2B) adenosine receptor antagonist at the time of infection increased MHC class II, CD86 and CD40 expression in L. amazonensis-infected DCs and restored the ability of the infected DCs to induce T-cell proliferation. Similar results were obtained through the inhibition of extracellular ATP hydrolysis using suramin. In conclusion, we propose that A(2B) receptor activation may be used by L. amazonensis to inhibit DC function and evade the immune response.

The androgen-regulated Calcium-Activated Nucleotidase 1 (CANT1) is commonly overexpressed in prostate cancer and is tumor-biologically relevant in vitro.

Previously, we identified the calcium-activated nucleotidase 1 (CANT1) transcript as up-regulated in prostate cancer. Now, we studied CANT1 protein expression in a large cohort of nearly 1000 prostatic tissue samples including normal tissue, prostatic intraepithelial neoplasia (PIN), primary carcinomas, metastases, and castrate-resistant carcinomas, and further investigated its functional relevance. CANT1 displayed predominantly a Golgi-type immunoreactivity with additional and variable cytoplasmic staining. In comparison to normal tissues, the staining intensity was significantly increased in PIN lesions and cancer. In cancer, high CANT1 levels were associated with a better prognosis, and castrate-resistant carcinomas commonly showed lower CANT1 levels than primary carcinomas. The functional role of CANT1 was investigated using RNA interference in two prostate cancer cell lines with abundant endogenous CANT1 protein. On CANT1 knockdown, a significantly diminished cell number and DNA synthesis rate, a cell cycle arrest in G(1) phase, and a strong decrease of cell transmigration rate and wound healing capacity of CANT1 knockdown cells was found. However, on forced CANT1 overexpression, cell proliferation and migration remained unchanged. In summary, CANT1 is commonly overexpressed in the vast majority of primary prostate carcinomas and in the precursor lesion PIN and may represent a novel prognostic biomarker. Moreover, this is the first study to demonstrate a functional involvement of CANT1 in tumor biology.

The influence of ecto-nucleotidases on Leishmania amazonensis infection and immune response in C57B/6 mice.

Previous results from our laboratory and from the literature have implicated the expression of ecto-nucleotidases in the establishment of Leishmania infection. In the present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed the immune response associated with the infection by these parasites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-gamma and have smaller parasite load, after 8weeks of infection. Furthermore, after 1week of infection, there is increased expression of the chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infection of peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role in infection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.

Ecto-nucleotidases, molecular properties and functional impact

Las ecto-nucleotidasas hidrolizan los nucleótidos extracelulares. Los nucleótidos se encuentran entre las sustancias mensajeras más ubicuas en vertebrados. Los receptores de nucleótidos se expresan en la superficie de prácticamente todas las células y muchas células expresan varios tipos de estos receptores. Se han identificado varias familias de ecto-nucleotidasas, las cuales difieren en su distribución tisular y en sus propiedades funcionales. Modulan la disponibilidad del ligando en los receptores de nucleótidos y de adenosina. Las ecto-nucleotidasas fueron identificadas por primera vez en la década de 1940. Los trabajos de las dos últimas décadas han mostrado sus características moleculares así como importantes propiedades funcionales. Utilizando delecciones génicas dirigidas se han mostrado claros ejemplos destacables de la importancia de las ecto-nucleotidasas en la señalización por nucléotidos y adenosina. Estos ejemplos abarcan desde el control del flujo sanguíneo y la angiogénesis a la modulación de las funciones inmunes y el desarrollo nervioso. Ecto-nucleotidasas específicas están asociadas con células madre en el cerebro adulto de mamífero, implicando un papel de los nucleótidos y nucleósidos en el control de la neurogénesis adulta. Las ecto-nucleotidasas representan importantes dianas terapéuticas para interferir en las vías de señalización mediadas por receptores P2 o P1. El desarrollo de ensayos de alto rendimiento promete una considerable aceleración en el desarrollo de inhibidores de subtipos específicos de ecto-nucleotidasas

Ecto-nucleotidases hydrolyze extracellular nucleotides. Nucleotides are amongst the most ubiquitous messenger substances in the vertebrate body. Receptors for nucleotides are expressed on the surface of essentially every cell and many cells carry several types of nucleotide receptors. Several families of ecto-nucleotidases have been identified that differ in tissue distribution and functional properties. They modulate ligand availability at nucleotide and adenosine receptors. Ectonucleotidases were first identified in the 1940ies. Work of the past two decades has unraveled molecular identities and important functional properties. Using targeted gene deletion clear examples highlighting the importance of ecto-nucleotidases in nucleotide and adenosine signaling have been elaborated. These reach from the control of blood flow and angiogenesis to the modulation of immune functions and neural development. Specific ecto-nucleotidases are associated with stem cells in the adult mammalian brain, implicating a role of nucleotides and nucleosides in the control of adult neurogenesis. Ecto-nucleotidases represent important therapeutic targets to interfere with P2 or P1 receptor-mediated receptor signaling pathways. The development of high throughput assays promises a considerable acceleration in the development of subtype-specific ecto-nucleotidase inhibitors

Effect of iron lactate overloading on adenine nucleotide levels and adenosine 3'-monophosphate forming enzyme in rat liver and spleen.

To elucidate the pathophysiological significance of adenosine 3'-monophosphate (3'-AMP) forming enzyme in rats, the effect of iron lactate overloading on the enzyme activities and adenine nucleotide levels in the liver and spleen was examined. Sprague-Dawley rats were fed a diet supplemented with 0%, 0.625% or 5.0% of iron lactate for 4 weeks. Iron deposition was found in periportal hepatocytes, Kupffer cells and macrophages of red pulp of the spleen. No significant changes in hematological parameters were detected. Although serum alkaline phosphatase and inorganic phosphorus levels elevated slightly in the 5.0% group, activities of alanine aminotransferase and aspartate aminotransferase, and levels of serum urea nitrogen and creatinine were not changed significantly. The ATP levels in the liver and spleen of iron fed groups were significantly decreased, but adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) levels were within control levels. On the other hand, the levels of ATP, ADP and AMP in the erythrocytes without mitochondria were not suppressed by the iron lactate overloading. Free activity of 3'-AMP forming enzyme, one of ribonucleases (RNase), was not changed in the liver of iron-overloaded rat, and total amount of 3'-AMP and adenosine formed after the treatment of the crude enzyme(s) with p-chloromercuribenzensulfonic acid, a SH blocker of RNase inhibitors, was decreased dose-dependently. On the contrary, free activity of 3'-AMP forming enzyme was enhanced dose-dependently in the spleen of iron-overloaded rat but the total activity was not changed. However, the free and total 3'-AMP forming enzyme activities in the liver and spleen of iron-overloaded rats became equal at the dosage of 5.0% of iron lactate. The results obtained suggested that iron loading might induce significant decrease in hepatic and splenic ATP levels via malfunction of their mitochondria and might lead dissociation of RNase-RNase inhibitor complex to activate 3'-AMP forming enzyme in both tissues.

Lithium inhibitable enzymes in postmortem brain of bipolar patients.

Despite considerable ongoing efforts at the epidemiological, genetic and molecular level, the etiology of bipolar disorder had not yet been elucidated. To study possible contributing components to the pathophysiology of this disorder, we have hypothesized that levels of enzymes inhibited by therapeutically relevant lithium ion concentrations in the brain of patients may differ from those in normal controls and may be involved in the etiology of the disorder. Three Li-inhibitable enzymes were studied in postmortem brain samples of bipolar patients and normal controls. The expression and function of the two enzymes that are obviously involved in signaling cascades, IMPase, involved in the second messenger system of the phosphatidylinositol cycle, and GSK-3, a mediator of an array of signaling cascades, were not found to be different in postmortem frontal and occipital cortex of bipolar patients and normal controls. Only PAP phosphatase protein levels, but not its mRNA levels or enzymatic activity, were found to be significantly decreased in frontal cortex of bipolar patients compared with normal controls.

Mammalian 5'(3')-deoxyribonucleotidase, cDNA cloning, and overexpression of the enzyme in Escherichia coli and mammalian cells.

5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.

Down regulation of gene expression by the vaccinia virus D10 protein.

Vaccinia virus genes are expressed in a sequential fashion, suggesting a role for negative as well as positive regulatory mechanisms. A potential down regulator of gene expression was mapped by transfection assays to vaccinia virus open reading frame D10, which encodes a protein with no previously known function. Inhibition was independent of the promoter type used for the reporter gene, indicating that the mechanism did not involve promoter sequence recognition. The inhibition was overcome, however, when the open reading frame of the reporter gene was preceded by the encephalomyocarditis virus internal ribosome entry site, which excludes the possibility of nonspecific metabolic or other antiviral effects and suggests that capped mRNAs or cap-dependent translation might be the target of the D10 product. The inducible overexpression of the D10 gene by a recombinant vaccinia virus severely inhibited viral protein synthesis, decreased the steady-state level of viral late mRNA, and blocked the formation of infectious virus.

Epidermal growth factor upregulates aminopeptidase N and 5'-nucleotidase in human glomerular mesangial cells.

Because epidermal growth factor (EGF) is likely to be released in the glomeruli during glomerular injury and mesangial cells possess specific receptors for EGF, we thought it to be of interest to examine whether this growth factor could influence the expression of ectoenzymes in cultured human mesangial cells. EGF stimulated 5'-nucleotidase and aminopeptidase N activities in intact human mesangial cells in a time- (24-72 h) and dose-dependent (0.1-50 ng ml(-1)) manner. Maximum stimulation represented 2.7- and 2-fold basal activities for 5'-nucleotidase and aminopeptidase N, respectively. EGF did not influence cyclic AMP production, and its effect on 5'-nucleotidase was additive to that of forskolin or 8-bromo-cAMP. In contrast, genistein (10 mg x ml(-1)), an inhibitor of tyrosine kinase, prevented EGF-dependent stimulation of aminopeptidase N and 5'-nucleotidase, suggesting that protein phosphorylation was involved in the signaling mechanism. EGF stimulated specifically the latter two enzymes since it had no effect on other ectoenzymes including alkaline phosphodiesterase I and Mg2+-ATPase activities. These results demonstrate that EGF, via the control of 5'-nucleotidase and aminopeptidase N, which are implied in adenosine formation and peptide processing, respectively, could play a role in human cultured mesangial cell contractility and proliferation.

The SAL1 gene of Arabidopsis, encoding an enzyme with 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast.

A cDNA library in a yeast expression vector was prepared from roots of Arabidopsis exposed to salt and was used to select Li(+)-tolerant yeast transformants. The cDNA SAL1 isolated from one of these transformants encodes a polypeptide of 353 amino acid residues. This protein is homologous to the HAL2 and CysQ phosphatases of yeast and Escherichia coli, respectively. Partial cDNA sequences in the data bases indicate that rice produces a phosphatase highly homologous to SAL1 and that a second gene homologous to SAL1 exists in Arabidopsis. The SAL1 protein expressed in E. coli showed 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities. In yeast, SAL1 restored the ability of a hal2/met22 mutant to grow on sulfate as a sole sulfur source, increased the intracellular Li+ tolerance, and modified Na+ and Li+ effluxes. We propose that the product of SAL1 participates in the sulfur assimilation pathway as well as in the phosphoinositide signaling pathway and that changes in the latter may affect Na+ and Li+ fluxes.

Na(+)-dependent amino acid uptake by human placental microvillous membrane vesicles: importance of storage conditions and preservation of cytoskeletal elements.

Human placental microvillous membrane vesicles (MMV) were purified by precipitation of non-microvillous membrane with MgCl2. Two aspects of MMV preparation were found to be important to the interpretation of amino acid transport studies: (1) storage conditions, and (2) preservation of cytoskeletal elements. In non-frozen MMV, MeAIB uptake was stimulated by an inward Na+ gradient and showed 'overshoot'. The initial Na(+)-dependent uptake rate was concentration-dependent with a Vmax of 640 +/- 80 pmol/mg/30 sec and a Km of 0.44 +/- 0.77 mM. Na(+)-stimulated cysteine uptake (65 +/- 23 pmol/mg/30 sec), previously thought to be very low or absent in the human placenta, was comparable to MeAIB, although there was no 'overshoot'. Cysteine uptake was partially stimulated by Li+. In general, freezing and storage at either -80 degrees C or -196 degrees C markedly reduced Na(+)-dependent uptake of several amino acids, compared to vesicles stored at 4 degrees C. The greatest reduction was seen with storage at -80 degrees C, especially with cysteine. There was no effect of storage temperature on Na(+)-independent amino acid uptake. For frozen vesicles, there was no difference in uptake for 12 versus 60 h storage. Removal of cytoskeletal proteins with the chaotropic agent, KSCN, resulted in greater enrichment of MMV marker proteins, but the preparation lost the capacity for active MeAIB uptake. These data, especially with regard to storage conditions, highlight the importance of precise definition of preparation and storage conditions when interpreting results of amino acid uptake by human placental MMV.

Crithidia luciliae: starvation for purines and/or phosphate leads to the enhanced surface expression of a protein responsible for 3'-nucleotidase/nuclease activity.

It has been shown previously that starvation of the trypanosomatid protozoan Crithidia luciliae for purines and/or inorganic phosphate results in increased levels of a surface membrane-associated 3'-nucleotidase/nuclease (3'-N'ase) activity which hydrolyzes both 3'-ribonucleotides and nucleic acids, thereby permitting the organisms to transport these essential nutrients across their cell membranes. A polypeptide with the requisite catalytic properties has been identified by an in situ gel activity assay following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In current studies, differential synthesis of the protein responsible for the 3'-N'ase activity was not demonstrable by comparisons of SDS-PAGE patterns of nutrient-replete or purine-starved parasites metabolically labeled with either [35S]methionine, [3H]leucine, or [3H]tyrosine. However, surface labeling of nutrient-replete and purine-starved cells revealed the enhanced expression of an 125I surface-labeled 43-kDa protein which comigrated with the 3'-N'ase activity in one- and two-dimensional electrophoretic systems. The amount of this surface-labeled peptide correlated with the level of 3'-N'ase activity as measured by test tube assay. Refeeding adenosine to purine-starved cells led to the loss of both the enzyme activity and the surface iodinatable 43-kDa band as a result of renewed cell division. Starvation of these organisms for phosphate also led to the enhanced expression of the 43-kDa radioiodinatable band. The results indicated that the 3'-N'ase protein, itself, is differentially expressed at the cell surface under conditions which lead to increased enzyme activity.

Leishmania donovani: regulated changes in the level of expression of the surface 3'-nucleotidase/nuclease.

Leishmania donovani promastigotes have previously been shown to possess a surface membrane bound 3'-nucleotidase/nuclease (3'-N'ase) capable of hydrolyzing both nucleic acids and 3'-ribonucleotides. The specific activity of the 3'-N'ase was increased following transfer of the parasites to fresh, nutrient-replete media or to media lacking purines and/or inorganic phosphate (Pi). In nutrient-replete media, the enzyme activity was transiently elevated during the lag and early logarithmic phases of the growth curve; enzyme activity fell as the cells continued into late log and stationary phases. Purine- and Pi-starved cells exhibited significantly greater levels of 3'-N'ase activity than nutrient-replete cells. These levels remained elevated as long as the organisms were maintained in the deficient media. Nutrient-replete and purine-starved 125I surface-labeled parasites displayed differences in electrophoretic patterns. Upon purine starvation, incorporation of radiolabel was increased in proteins which migrated with apparent molecular weights of 70, 43, and 40 kDa. Comigration, in both one- and two-dimensional systems, of 3'-N'ase activity with the radiolabeled 43-kDa band demonstrated that this band was the catalytically active protein. Peptide mapping of the 70-, 43-, and 40-kDa proteins failed to demonstrate similarities in peptide sequence consistent with either a degradation or a precursor/product relationship. Treatment of the 43- and 40-kDa peptides with N-Glycanase indicated that they were differentially glycosylated. The cumulative results of these studies indicated that L. donovani can respond to altered culture conditions by the differential expression of surface proteins. In particular, the differential expression of the protein responsible for 3'-N'ase activity is consistent with the role of this enzyme in purine acquisition.

An inducible 3'-nucleotidase/nuclease from the trypanosomatid Crithidia luciliae. Purification and characterization.

Several species of protozoan parasites of the family Trypanosomatidae have a surface membrane-associated enzyme which is capable of hydrolyzing extracellular 3'-nucleotides and nucleic acids, thereby aiding in the acquisition of nutritionally required purines and Pi from their hosts. In Crithidia luciliae, this 3'-nucleotidase/nuclease previously has been shown to be highly regulated as purine and/or Pi starvation of this trypanosomatid leads to as much as a 1000-fold increase in enzyme activity. We have purified the enzyme to apparent homogeneity from detergent extracts of purine-starved C. luciliae by heparin-agarose chromatography followed by Mono Q and Mono S fast protein liquid chromatography. The enzyme had an apparent molecular weight of 43,000 and a pI of approximately 5.8. The enzyme displayed broad pH optima, with peaks at 8.0, for both nucleotidase and nuclease activities. The pH optima shifted to lower values when the activity was assayed in the presence of sulfhydryl reagents. The enzyme was most active with 3'-AMP and poly(A) in nucleotidase and nuclease assays, respectively. As a nuclease the enzyme hydrolyzed RNA at a faster rate than single-stranded DNA with no detectable hydrolysis of double-stranded DNA. The loss of enzyme activity which occurred upon storage at acid pH was prevented by the inclusion of Zn2+ in storage buffers. The physicochemical and kinetic properties of this trypanosomatid enzyme suggest that it is similar to the class I nucleases found in fungi and in germinating seedlings of higher plants.

Cloning and expression of the 5'-nucleotidase gene of Vibrio parahaemolyticus in Escherichia coli and overproduction of the enzyme.

The gene encoding the membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus was cloned and expressed in Escherichia coli. Cells of E. coli harboring a plasmid, pNUT5, which carries the 5'-nucleotidase gene were able to grow on ATP as the sole source of carbon, although the original cells were not. The 5'-nucleotidase activity was detected in whole cells of E. coli harboring pNUT5 and in membrane vesicles prepared from these cells. Most properties of the 5'-nucleotidase produced in E. coli, that is, its requirements for Cl- and Mg2+, substrate specificity, and inhibition by Zn2+, were similar to those observed in V. parahaemolyticus, but some alterations in properties were observed: The 5'-nucleotidase was partially inducible in V. parahaemolyticus, but its expression in E. coli was completely constitutive. The specific activity of the 5'-nucleotidase in membrane vesicles of E. coli harboring the plasmid was 30 times that observed in whole cells, whereas the specific activities in membrane vesicles and in whole cells of V. parahaemolyticus were almost the same. A new, dense band of protein with an apparent molecular mass of 63 kDa was detected when membrane proteins of E. coli harboring the plasmid were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Induction of adenosine deaminase and 5' nucleotidase activity in cultured human blood monocytes and monocytic leukemia (THP-1) cells by differentiating agents.

The in vitro effect of short-term culture as well as the effect of retinol (ROH), retinoic acid (RA), muramyl dipeptide [( Abu']MDP), lipopolysaccharide (LPS), and gamma interferon (IFN-gamma) on the induction of the purine metabolic enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and 5'nucleotidase (5NT) in human peripheral blood monocytes (HPBM) was examined. HPBM isolated by centrifugal elutriation were cultured for up to 96 h. Following an initial time lag of 24 h, mean ADA activity from seven separate experiments as measured in nmoles/10(6) cells/h increased from a baseline of 31.3 +/- 9.3 to 57.8 +/- 16.4 (P less than 0.005) at 72 h and to 72 +/- 21.5 (P less than .025) by 96 h. 5NT activity increased from a baseline of 2.2 +/- 0.9 to a maximum of 44 +/- 10.1 by 72 h and then declined to 29 +/- 18 (P less than 0.005) by 96 h, while no significant change in PNP activity was observed. HPBM incubated for 3 d with optimal concentrations of LPS, RA, and IFN-gamma had increases in ADA and 5NT activity ranging from three- to 10-fold compared to HPBM cultured in media alone, whereas no effect was observed with ROH and [Abu']MDP. RA, but not ROH, significantly enhanced ADA activity in a monocytic leukemia cell (THP-1) line. Addition of RA or the tumor promoter, phorbol 12-myristic 13-acetate (PMA), to HPBM or THP-1 cells resulted in significant increases in 5NT activity with opposite effects on ADA activity. These findings suggest that the biological mechanisms associated with differentiation in normal and malignant monocytes seem to be related and that the sequence and degree to which the various differentiation agents induce the enzyme elevations are also related to the mechanisms of activation/differentiation.

The effect of sorbose on NAD(P)ase production by Aspergillus nidulans.

1. NAD(P)ase activity was stimulated when 1% sorbose was present in the culture medium of A. nidulans, and this effect was partially reversed by 1% glucose. 2. The level of extracellular NAD(P)ase was more affected by sorbose in the culture medium than the intracellular enzyme and no morphological changes were obtained. 3. The sorbose effect on NAD(P)ase activity appears to be specific since two other exoenzymes tested (beta-glucosidase and alkaline protease) show normal secretion patterns. 4. These findings suggest that the sorbose effect on NAD(P)ase production may be the consequence of metabolic disorders not necessarily linked with the morphological changes induced by the ketohexose.

Properties of the membrane-bound 5'-nucleotidase and utilization of extracellular ATP in Vibrio parahaemolyticus.

Vibrio parahaemolyticus utilized ATP, ADP or AMP as the sole source of carbon. About three times higher activity of membrane-bound 5'-nucleotidase was observed in cells grown in the presence of these nucleotides than in their absence: and therefore the enzyme seems to be inducible. Since the 5'-nucleotidase activity could be measured with whole cells, the active site of this enzyme appears to be outwardly oriented. Both Mg2+ and Cl- were required for activity. Among the divalent cations tested, Mn2+ and Co2+ could replace Mg2+ to some extent, whereas Zn2+ strongly inhibited activity. Among the anions tested, Br-, I- and NO3- could replace Cl-, but SO4(2-) and CH3COO- could not. When cells were grown with ATP, Cl- was indispensable and Zn2+ strongly inhibited growth. Therefore, it is concluded that extracellular ATP and other 5'-nucleotides are cleaved by the membrane-bound 5'-nucleotidase outside the cells and that the adenosine produced is then utilized.

Characterization of Pi-repressible enzymes secreted in culture media by Neurospora crassa wild-type cells and null-type mutants.

In wild-type mycelial cultures of Neurospora crassa under Pi-limited conditions, alkaline phosphatase, cyclic phosphodiesterases I, II, III, and IV, 5'-nucleotidase, acid and alkaline nucleases, RNase N1, and a newly detected endonuclease were secreted into the culture media. These enzymes were either not produced or were produced in very reduced levels in mutants nuc-1, -2, -3, -4, -5, -6, and -7 and cpd-4. The proteins were examined by polyacrylamide gel electrophoresis in a manner which allowed the identification of each of them.

The synthesis and turnover of 5'-nucleotidase in primary cultured hepatocytes.

The synthesis and degradation of 5'-nucleotidase has been studied in rat hepatocytes. Primary cultures of rat hepatocytes were established with the cells showing evidence of polarity after 24-36 h in culture. After a 30 h lag period 5'-nucleotidase activity increased to a plateau level similar to the activity found in whole liver. The half life of the enzyme after reaching the plateau of activity was 22.8 h. Pulse-chase biosynthetic labelling studies of 5'-nucleotidase in the cultured hepatocytes using [35S]methionine showed that the 5'-nucleotidase monomer was synthesised as an Mr 67,000 form which was converted to the mature Mr 72,000 form. [35S]Methionine labelling studies in the presence of tunicamycin showed that the unglycosylated protein monomer was an Mr 57,000 form. The immature Mr 67,000 form of 5'-nucleotidase was sensitive to endoglycosidase H, whereas the mature form was sensitive only to endoglycosidase F. The data presented are consistent with 5'-nucleotidase in a polarised cell being synthesised and processed like other membrane glycoproteins, in contrast to earlier reports.