5'-Nucleotidase activity of mouse peritoneal macrophages. I. Synthesis and degradation in resident and inflammatory populations.
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Mouse resident peritoneal macrophages display sufficient 5'-nucleotidase activity to hydrolyze 58 nm AMP/min per cell protein. This activity increases approximately 163 nm AMP/min per mg after 72 h in culture. The enzyme is renewed in unstimulated cells with a half-time of 13.9 h. The activity is not reduced by treatment of intact cells with a variety of proteolytic enzymes, including trypsin, pronase, urokinase, and plasmin. Cells obtained from an inflammatory exudate have diminished or absent levels of enzyme activity. Endotoxin-elicited cells display enzyme activitiy of 20.9 nm AMP/min per mg, while thioglycollate-stimulated macrophages have no detectable activity. The reduced level of activity in endotoxin-stimulated cells is due to their elevated rate of enzyme degradation, with a half-time of 6.9 h. Their rate of enzyme synthesis is essentially normal. No evidence for latent enzyme activity could be obtained in thioglycollate-stimulated cells, nor do these cells produce any inhibition of normal cell enzyme activity. Serum deprivation reduces the enzyme activity of resident cells to about 45% of control activity. These conditions do not significantly affect the rate of enzyme synthesis, but again are explainable by an increase in the rate of enzyme degradation. Pinocytic rate is elevated in endotoxin-stimulated cells which show a more rapid rate of enzyme degradation than unstimulated cells do. However, in serum-free conditions, the rate of enzyme degradation is doubled with no change in the pinocytic rate of the cells. (+info)
5'-Nucleotidase activity of mouse peritoneal macrophages. II. Cellular distribution and effects of endocytosis.
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The diazonium salt of sulfanilic acid (DASA) can inactivate about 80% of the total 5'-nucleotidase of viable macrophages. The remaining 20% can be inactivated if the cells are first lysed in detergent, and presumably represents an intracellular pool of 5'-nucleotidase. The bulk of this pool may represent cytoplasmic vesicles derived from plasma membrane by endocytosis. This internal compartment is expanded up to threefold immediately after the cells have ingested a large latex load. This is consistent with previous observations on the internalization of 5'-nucleotidase in latex phagosomes. In latex-filled cells this intracellular pool of enzyme is inactivated over a few hours, and the cells then slowly increase their enzyme activity to nearly normal levels. However, 24 h after latex ingestion the metabolism of 5'-nucleotidase in these recovered cells is abnormal, as the rate of enzyme degradation is about twice the normal rate, and the DASA-insensitive enzyme pool in these cells is strikingly diminished. This may reflect effects of the accumulated indigestible particles on the fate of incoming pinocytic vesicles or on newly synthesized plasma membrane precursor. Another endocytic stimulus, concanavalin A, also reduces the total cell 5'-nucleotidase activity. This effect, which is time and temperature dependent, can be prevented by the competitive sugar alpha-methyl mannose. The concanavalin A inhibition can be reversed in the absence of new protein synthesis or in cells cultivated in serum-free conditions. It is not known whether the effect of concanavalin A on 5'-nucleotidase depends upon the interiorizaiton of plasma membrane or is strictly associated with events at the cell surface. (+info)
Cloning and characterization of a mammalian lithium-sensitive bisphosphate 3'-nucleotidase inhibited by inositol 1,4-bisphosphate.
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Discovery of a structurally conserved metal-dependent lithium-inhibited phosphomonoesterase protein family has identified several potential cellular targets of lithium as used to treat manic depression. Here we describe identification of a novel family member using a "computer cloning" strategy. Human and murine cDNA clones encoded proteins sharing 92% identity and were highly expressed in kidney. Native and recombinant protein harbored intrinsic magnesium-dependent bisphosphate nucleotidase activity (BPntase), which removed the 3'-phosphate from 3'-5' bisphosphate nucleosides and 3'-phosphoadenosine 5'-phosphosulfate with Km and Vmax values of 0.5 microM and 40 micromol/min/mg. Lithium uncompetitively inhibited activity with a Ki of 157 microM. Interestingly, BPntase was competitively inhibited by inositol 1,4-bisphosphate with a Ki of 15 microM. Expression of mammalian BPntase complemented defects in hal2/met22 mutant yeast. These data suggest that BPntase's physiologic role in nucleotide metabolism may be regulated by inositol signaling pathways. The presence of high levels of BPntase in the kidney are provocative in light of the roles of bisphosphorylated nucleotides in regulating salt tolerance, sulfur assimilation, detoxification, and lithium toxicity. We propose that inhibition of human BPntase may account for lithium-induced nephrotoxicity, which may be overcome by supplementation of current therapeutic regimes with inhibitors of nucleotide biosynthesis, such as methionine. (+info)
A nod factor binding lectin with apyrase activity from legume roots.
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A lectin isolated from the roots of the legume, Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date. This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands. This lectin-nucleotide phosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes. LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia. These properties suggest that this protein may play a role in the rhizobium-legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant. (+info)
Reduced purine 5'-nucleotidase activity in lymphocytes of patients with systemic lupus erythematosus: results of a pilot study.
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OBJECTIVE: To investigate purine metabolism in patients with systemic lupus erythematosus (SLE) for possible abnormalities that might be related to their overall impaired immune function. METHODS: This pilot study included 17 patients with SLE (2 men, 15 women). Enzyme activities of the purine enzymes 5'-nucleotidase (5'NT), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) were measured in peripheral blood mononuclear cells (PBMC) and also in fractions of T cells (differentiation antigen CD3+) (n = 12) and B cells (CD19+) (n = 9). The activity of the thiopurine enzyme thiopurine-methyltransferase (TPMT) was measured in red cell lysate. Routine blood tests and indices of disease activity were measured as well. Results were compared with those of healthy volunteers. RESULTS: Compared with their controls, the female SLE patients had a more than 50% reduced activity of 5'NT in the T cell fraction (p = 0.001) and in PBMC (p < 0.000). 5'NT activity was also lower in B cells, but this was not statistically significant. Enzyme activities did not correlate with indices of disease activity, disease duration or the B cell/T cell ratio and no influence of medication was found. CONCLUSION: Reduced lymphocyte 5'NT activity is a novel finding in SLE. These results indicate that purine metabolism in SLE may be disturbed. Consequences of a low 5'NT activity may be an intracellular accumulation of (deoxy)purine nucleotides and a reduction of adenosine production. It is hypothesised that these factors may play a part in the overall impaired immune function and in the chronicity of inflammation in SLE. (+info)
Biochemical and genetic analyses of the role of yeast casein kinase 2 in salt tolerance.
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Saccharomyces cerevisiae cells lacking the regulatory subunit of casein kinase 2 (CK-2), encoded by the gene CKB1, display a phenotype of hypersensitivity to Na(+) and Li(+) cations. The sensitivity of a strain lacking ckb1 is higher than that of a calcineurin mutant and similar to that of a strain lacking HAL3, the regulatory subunit of the Ppz1 protein phosphatase. Genetic analysis indicated that Ckb1 participates in regulatory pathways different from that of Ppz1 or calcineurin. Deletion of CKB1 increased the salt sensitivity of a strain lacking Ena1 ATPase, the major determinant for sodium efflux, suggesting that the function of the kinase is not mediated by Ena1. Consistently, ckb1 mutants did not show an altered cation efflux. The function of Ckb1 was independent of the TRK system, which is responsible for discrimination of potassium and sodium entry, and in the absence of the kinase regulatory subunit, the influx of sodium was essentially normal. Therefore, the salt sensitivity of a ckb1 mutant cannot be attributed to defects in the fluxes of sodium. In fact, in these cells, both the intracellular content and the cytoplasm/vacuole ratio for sodium were similar to those features of wild-type cells. The possible causes for the salt sensitivity phenotype of casein kinase mutants are discussed in the light of these findings. (+info)
A yeast Golgi E-type ATPase with an unusual membrane topology.
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E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+). The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized. (+info)
Evidence for the translocation of 5'-nucleotidase across hepatic membranes in vivo.
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Hepatic 5'-nucleotidase (EC 3.1.3.5; 5'-ribonucleotide phosphohydrolase) activity has been studied in cisternal elements of the Golgi complex and in secretion vacuoles, both isolated after ethanol administration to rats in vivo. The enzyme in secretion vacuoles was latent, so that a 5-fold increase in activity was observed when incubations were carried out in the presence of detergent; evidence is presented that the latency is caused by the impermeability of the membrane to substrate. Essentially no latency was observed in Golgi cisternae. Confirming the results of Farquhar et al. [(1974) J. Cell Biol. 60, 8-25], reaction product from 5'-nucleotidase was localized by cytochemical procedures on the inside of secretion vacuoles and on the cytoplasmic side of Golgi cisternae. After solubilization in detergent, the enzyme from both fractions reacted almost identically with both antibody to the purified enzyme and concanavalin A. In contrast, when intact fractions were incubated with an excess of antibody or concanavalin A, only 22-23% of the enzyme was inhibited in secretion vacuoles whereas 51-84% was inhibited in Golgi cisternae. Sonication of secretion vacuoles in the presence of antibody or concanavalin A increased the inhibition 2- to 3-fold. It is suggested that during the formation of secretion vacuoles from the Golgi cisternae, 5'-nucleotidase is translocated from the cytoplasmic side of the membrane to the inside. (+info)