Journal articles: 'Ectoenzyme activities'

1

Ammerman, JW, and WB Glover. "Continuous underway measurement of microbial ectoenzyme activities in aquatic ecosystems." Marine Ecology Progress Series 201 (2000): 1–12. http://dx.doi.org/10.3354/meps201001.

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Orcutt, KM, K. Gundersen, and JW Ammerman. "Intense ectoenzyme activities associated with Trichodesmium colonies in the Sargasso Sea." Marine Ecology Progress Series 478 (March 25, 2013): 101–13. http://dx.doi.org/10.3354/meps10153.

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3

Sala, M. M., and H. Güde. "Development of microbial ectoenzyme activities in littoral sediments of Lake Constance." SIL Proceedings, 1922-2010 25, no. 1 (September 1993): 633. http://dx.doi.org/10.1080/03680770.1992.11900211.

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4

Chen, X., and J. D. Catravas. "PMA-activated neutrophils decrease pulmonary endothelial ectoenzyme activities in perfused rabbit lungs." American Journal of Physiology-Lung Cellular and Molecular Physiology 263, no. 6 (December 1, 1992): L650—L656. http://dx.doi.org/10.1152/ajplung.1992.263.6.l650.

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We have studied the effects of phorbol 12-myristate 13-acetate (PMA, 15 micrograms) on pulmonary endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] function in isolated rabbit lungs perfused in situ with platelet-poor (PPP) or platelet-rich (PRP) plasma in the presence or absence of neutrophils. Enzyme activities were estimated from the hydrolysis of the substrates [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) by ACE and 14C-labeled AMP by NCT during a single transpulmonary passage, using indicator-dilution techniques. In all treatment groups PMA produced a delayed increase in pulmonary vascular resistance to about three times the control value. PMA alone [in lungs perfused with PPP (n = 5 animals) or PRP (n = 6)] or neutrophils alone (in PPP-perfused lungs, n = 5) had no effect on enzyme activity. However, PMA-activated neutrophils (n = 5) decreased percent metabolism (%M) of [3H]BPAP from 87 +/- 3 to 77 +/- 4% (30 min after PMA), and the apparent first-order parameter [ratio of maximum activity to Michaelis constant (Amax/Km)] for ACE from 821 +/- 114 to 613 +/- 61 ml/min (30 min after PMA). At the same time, Km values of BPAP for ACE and AMP for NCT were elevated from 9.2 +/- 2.2 to 19.3 +/- 3 microM and 6.7 +/- 1.2 to 15.1 +/- 3.6 microM, respectively, whereas Amax (product of enzyme mass and rate of product formation, thus an index of perfused microvascular surface area) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

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5

Boyle, J. M., Y. Hey, and M. Fox. "Nucleotide ectoenzyme activities of human and Chinese hamster fibroblasts in tissue culture." Biochemical Genetics 27, no. 11-12 (December 1989): 655–71. http://dx.doi.org/10.1007/bf02396058.

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6

Chen, X., M. Tzanela, M. K. Baumgartner, J. R. McCormick, and J. D. Catravas. "PMA-activated neutrophils decrease ectoenzyme activities in rabbit aortic endothelial cells in culture." American Journal of Physiology-Lung Cellular and Molecular Physiology 263, no. 6 (December 1, 1992): L657—L663. http://dx.doi.org/10.1152/ajplung.1992.263.6.l657.

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We have studied the effects of phorbol 12-myristate 13-acetate (PMA)-activated neutrophils [polymorphonuclear leukocytes (PMN)] on endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] activities in cultured rabbit aortic endothelial cells (EC) with the use of [3H]benzoyl-Phe-Ala-Pro and 14C-labeled AMP as substrates, respectively, under first-order reaction conditions. PMA (1–1,000 ng/ml) or PMN alone had no effect on ACE activity. When PMA was incubated together with PMN (PMN/EC = 1.25:1 or 2.5 x 10(5) neutrophils/ml) for 4 h in Earle's salts, a PMA dose-dependent decrease in ACE activity was observed. Threshold PMA concentration was 2 ng/ml. At 8 ng PMA/ml, ACE activity was totally abolished, without any evidence of cytotoxicity, as inferred from release of 51Cr from prelabeled EC. The decrease in ACE activity was also dependent on PMN concentration and was detectable at PMN/EC values as low as 1.25:10 (0.25 x 10(5) PMN/ml). Inhibition of ACE occurred as early as 1 h after incubation (PMA 10 ng/ml, PMN/EC = 1.25:1). PMA alone caused a small but significant increase in NCT activity, whereas PMA coincubation with PMN produced a significant decrease in NCT activity (20–29%), which was PMA and PMN concentration independent. PMA increased PMN adherence to endothelial monolayers in a concentration-dependent manner. Pretreating PMN with monoclonal antibody 60.3 (raised against the adhesion glycoprotein CD18) or placing a 2-microns filter between PMN and EC, protected the decrease in ACE activity.(ABSTRACT TRUNCATED AT 250 WORDS)

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7

Morell, Andreas, Gabriele Losa, Stephan Carrel, Didier Heumann, and Vladimir E. Von Fliedner. "Determination of ectoenzyme activities in leukemic cells and in established hematopoietic cell lines." American Journal of Hematology 21, no. 3 (March 1986): 289–98. http://dx.doi.org/10.1002/ajh.2830210308.

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8

Papapetropoulos, A., L. A. Elmore, and J. D. Catravas. "Relationship between volume of bathing medium and ectoenzyme activity in monolayers of cultured BPAEC." American Journal of Physiology-Lung Cellular and Molecular Physiology 271, no. 3 (September 1, 1996): L464—L469. http://dx.doi.org/10.1152/ajplung.1996.271.3.l464.

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It is unclear whether all or a fraction of the capillary plasma volume (Vcp) serves as the reaction volume (Vr) for pulmonary capillary endothelial ectoenzymes, in vivo. Cultured endothelial cell (EC) monolayers provide a convenient model for studying EC-bound enzyme-Vr relationships. Because the Michaelis-Menten parameter [maximum velocity of enzyme reaction (Vmax) = E x kcat/Vr, where E is enzyme mass and kcat is the constant of product formation] is inversely proportional to Vr, we hypothesized that increasing the volume of medium (Vm) bathing EC monolayers would proportionally reduce the calculated Vmax (or Vmax/K(m), where K(m) is the Michaelis constant) values of an ectoenzyme reacting with a substrate only if, and as long as, Vm = Vr. To test this hypothesis, studies were performed in bovine pulmonary arterial EC grown to confluence. Activities of angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT) were assayed in Earle's balanced salts solution utilizing [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) and 5'-[14C]AMP as substrates, respectively. Under first-order reaction conditions and at constant substrate concentrations ([BPAP] = 15 nM, [AMP] = 1 microM), Vmax/K(m) ratios of ACE and NCT declined to 20% of their original values, as Vm increased from 0.6 to 2 ml. ACE activity was also studied at constant substrate mass (BPAP = 7 pmol) under first-order reaction conditions. Again, enzyme activity (Vmax/K(m)) declined proportionally to increasing Vm. Under zero-order reaction conditions ([BPAP] = 250 microM), ACE activity (Vmax) was similarly related to Vm. Linear regression analyses revealed that ACE or NCT would recognize up to at least 3 ml Vm, a volume vastly exceeding that of Vcp in a section of the capillary bed composed of an equivalent number of ECs, thus suggesting that Vcp could serve as the reaction volume for pulmonary capillary EC ectoenzymes in vivo.

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9

Fukuda, Rumi, Yoshiki Sohrin, Nobue Saotome, Hideki Fukuda, Toshi Nagata, and Isao Koike. "East-west gradient in ectoenzyme activities in the subarctic Pacific: Possible regulation by zinc." Limnology and Oceanography 45, no. 4 (June 2000): 930–39. http://dx.doi.org/10.4319/lo.2000.45.4.0930.

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10

Sala, MM, M. Karner, L. Arin, and C. Marrasé. "Measurement of ectoenzyme activities as an indication of inorganic nutrient imbalance in microbial communities." Aquatic Microbial Ecology 23 (2001): 301–11. http://dx.doi.org/10.3354/ame023301.

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11

Donachie, Stuart P., James R. Christian, and David M. Karl. "Nutrient regulation of bacterial production and ectoenzyme activities in the subtropical North Pacific Ocean." Deep Sea Research Part II: Topical Studies in Oceanography 48, no. 8-9 (January 2001): 1719–32. http://dx.doi.org/10.1016/s0967-0645(00)00158-2.

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12

DE FLORA, Antonio, Lucrezia GUIDA, Luisa FRANCO, Elena ZOCCHI, Mario PESTARINO, Cesare USAI, Carla MARCHETTI, Ernesto FEDELE, Giovanni FONTANA, and Maurizio RAITERI. "Ectocellular in vitro and in vivo metabolism of cADP-ribose in cerebellum." Biochemical Journal 320, no. 2 (December 1, 1996): 665–71. http://dx.doi.org/10.1042/bj3200665.

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CD38, a type II transmembrane glycoprotein predominantly expressed in blood cells, is a bifunctional ectoenzyme directly involved in the metabolism of cADP-ribose (cADPR). This is a potent Ca2+ mobilizer in several types of cells. The relationship between the ectocellular site of cADPR production and its intracellular calcium-related functions is poorly understood. Cultured rat cerebellar granule cells showed both enzymic activities of CD38, ADP-ribosyl cyclase and cADPR hydrolase, at a ratio of 16 to 1 respectively, and were immunostained by the anti-(human CD38) monoclonal antibody IB4. In these cells externally added cADPR and β-NAD+ (the precursor of cADPR), but not α-NAD+ or ADP-ribose, enhanced the peak of the depolarization-induced rise in intracellular Ca2+ concentration. This effect was inhibited by 1 µM ryanodine, suggesting a potentiation of calcium-induced calcium release by cADPR. CD38 ectoenzyme activities, ADP-ribosyl cyclase and cADPR hydrolase, were also demonstrated in vivo by microdialysis of adult rat cerebellum, where IB4 bound to granule neurons selectively. Trace amounts (11.5±3.8 nM) of NAD+ were detected by microdialysis sampling and sensitive assays in the basal interstitial fluid of the cerebellum. These results provide a link between ectocellular cADPR turnover and intracellular calcium mobilization in cerebellum.

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13

Jaeger, Stephanie A., Brian M. Gaas, Gary P. Klinkhammer, and James W. Ammerman. "Multiple Enzyme Analyzer (MEA): Steps toward the in situ detection of microbial community ectoenzyme activities." Limnology and Oceanography: Methods 7, no. 11 (October 23, 2009): 716–29. http://dx.doi.org/10.4319/lom.2009.7.716.

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14

Christian, JR, and DM Karl. "Measuring bacterial ectoenzyme activities in marine waters using mercuric chloride as a preservative and a control." Marine Ecology Progress Series 123 (1995): 217–24. http://dx.doi.org/10.3354/meps123217.

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15

Gatmaitan, Z. C., A. T. Nies, and I. M. Arias. "Regulation and translocation of ATP-dependent apical membrane proteins in rat liver." American Journal of Physiology-Gastrointestinal and Liver Physiology 272, no. 5 (May 1, 1997): G1041—G1049. http://dx.doi.org/10.1152/ajpgi.1997.272.5.g1041.

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The bile canalicular membrane contains four specific ATP-dependent transport processes that are involved in secretion of bile acids, non-bile acid organic anions (mrp1), phospholipids (mdr2), and organic cations (mdr3). The aim of this study was to determine how the canalicular presence of these transport proteins is regulated. Canalicular membrane vesicles (CMV) were prepared from livers of rats treated with taurocholate (TC) and/or dibutyryl-adenosine 3',5'-cycle monophosphate (DBcAMP) with and without pretreatment with colchicine. Transport studies were performed with radiolabeled substrates. Changes in the relative amounts of transport proteins were determined by Western blots. Compared with controls, the specific activity of each of the transport processes was enhanced 1.5- and 3-fold with TC and DBcAMP treatments, respectively. Western blots revealed the same increases with mdr2 and mdr3. Pretreatment of rats with colchicine prevented these responses fully with TC treatment, whereas only partial prevention was obtained with DBcAMP treatment. Besides the ATP-dependent transporters, the relative specific activities of the canalicular membrane ectoenzyme markers, leucine aminopeptidase and gamma-glutamyltranspeptidase, were also affected the same way. These results suggest that TC and DBcAMP increase the specific activity of the canalicular ATP-dependent transport proteins and some canalicular membrane ectoenzymes by stimulating an increase in the relative amounts of these proteins in the membrane.

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16

Park, JS, DH Choi, CY Hwang, GJ Park, and BC Cho. "Seasonal study on ectoenzyme activities, carbohydrate concentrations, prokaryotic abundance and production in a solar saltern in Korea." Aquatic Microbial Ecology 43 (June 27, 2006): 153–63. http://dx.doi.org/10.3354/ame043153.

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17

Hara-Yokoyama, Miki, Mutsuko Kukimoto-Niino, Kazue Terasawa, Satoru Harumiya, Katarzyna A. Podyma-Inoue, Nobumasa Hino, Kensaku Sakamoto, et al. "Tetrameric Interaction of the Ectoenzyme CD38 on the Cell Surface Enables Its Catalytic and Raft-Association Activities." Structure 20, no. 9 (September 2012): 1585–95. http://dx.doi.org/10.1016/j.str.2012.06.017.

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18

D'Alesandro, M., M. Patchen, and B. Solberg. "Effects of particulate glucan on murine peritoneal macrophage 5'-nucleotidase leucine aminopeptidase and alkaline phosphodiesterase ectoenzyme activities." International Journal of Immunopharmacology 7, no. 3 (January 1985): 304. http://dx.doi.org/10.1016/0192-0561(85)90191-2.

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19

Hooper, N. M., J. Hryszko, and A. J. Turner. "Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme." Biochemical Journal 267, no. 2 (April 15, 1990): 509–15. http://dx.doi.org/10.1042/bj2670509.

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Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.

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20

Dvorak, A. M., R. A. Monahan-Earley, H. F. Dvorak, and S. J. Galli. "Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils." Journal of Histochemistry & Cytochemistry 35, no. 3 (March 1987): 351–60. http://dx.doi.org/10.1177/35.3.3819377.

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We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.

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21

Riemann, Lasse, Grieg F. Steward, and Farooq Azam. "Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom." Applied and Environmental Microbiology 66, no. 2 (February 1, 2000): 578–87. http://dx.doi.org/10.1128/aem.66.2.578-587.2000.

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ABSTRACT Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment (≈24 μg of chlorophylla liter−1). At this time bacterial abundance abruptly decreased from 2.8 × 106 to 0.75 × 106 ml−1, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- andCytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.

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22

Deaglio, Silvia, Tiziana Vaisitti, Semra Aydin, Luciana Bergui, Davide Rossi, Lisa Bonello, Giovanna Chiorino, Gianluca Gaidano, and Fabio Malavasi. "CD38 modulates CXCL12-mediated chemotaxis: a novel mechanism in homing of chronic lymphocytic leukemia cells (98.7)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 98.7. http://dx.doi.org/10.4049/jimmunol.182.supp.98.7.

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Abstract Human CD38, an ectoenzyme involved in the catabolism of NAD, can also bind the non-substrate ligand CD31, resulting in activation and proliferation of distinct lymphocyte populations. CD38 is clinically used as a negative prognostic marker for chronic lymphcytic leukemia (CLL) patients. We adopted CLL as a model to study the relevance of CD38 in vivo. CD38 signals trigger proliferation and increase survival of CLL cells. Further, the simultaneous expression of CD38 and ZAP-70 defines a genetically distinct subset of CLL cells with heightened responses to CXCL12, a critical chemokine in CLL homing from blood to lymphoid organs. Here we show that CD38 is directly involved in CXCL12-mediated homing. CD38 signals facilitate short- (ERK1/2 phosphorylation) and long-term (chemotaxis) effects induced by CXCL12. Assays using i) a specific inhibitor of CD38 enzymatic activities, ii) agonistic mAbs, and iii) microarray technology indicate that increased homing depends on the receptor functions of CD38. Conversely, blocking anti-CD38 mAbs significantly weaken CXCL12 responses. This is due to physical association on the membrane between CD38 and CXCR4 (the CXCL12 receptor), as shown by i) co-immunoprecipitation, ii) confocal microscopy and iii) measurement of CXCL12 binding to its receptor. These results indicate that CD38 modulates CXCL12 signals, representing a novel mechanism in CLL chemotaxis and homing.

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23

Beaudoin, A. R., J. Sevigny, G. Grondin, S. Daoud, and F. P. Levesque. "Purification, characterization, and localization of two ATP diphosphohydrolase isoforms in bovine heart." American Journal of Physiology-Heart and Circulatory Physiology 273, no. 2 (August 1, 1997): H673—H681. http://dx.doi.org/10.1152/ajpheart.1997.273.2.h673.

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Two ATP diphosphohydrolase (ATPDase) isoforms have been purified from the bovine heart ventricle. The purification procedure includes the following steps: differential centrifugation, sucrose cushion centrifugation, solubilization with Triton X-100, DEAE agarose ion exchange, and Affi-Gel blue-Sepharose and concanavalin A (con A)-Sepharose chromatographies. The purified enzyme has an optimum pH of catalysis of 7.5 and requires Ca2+ or Mg2+. The apparent Michaelis constant of the enzyme, with ADP as the substrate, is 29 microM, and the apparent maximal velocity is 1.6 mumol.min-1.mg protein-1. Substrate specificity, heat-inactivation curves, and copurification of adenosinetriphosphatase (ATPase) and adenosinediphosphatase (ADPase) activities confirmed the identity of the purified enzyme as an ATPDase. In addition, polyacrylamide gel electrophoresis, under nondenaturing conditions, showed identical migration patterns for the protein involved in ATPase and ADPase activities. Western blot analysis, with an antibody that specifically recognizes the NH2-terminal sequence of pig pancreas ATPDase and specifically reacts with bovine and human ATPDases, showed cross-reactivity with the purified ATPDase isoforms from the bovine heart. Immunocytochemical localization in the ventricle produced strong reactions with the plasma membrane of Purkinje fiber cells and the majority of myocardial cells. Immunoreactivity was variable, producing a mosaic-like aspect. As expected, smooth muscle cells and endothelial cells of coronary vessels were highly reactive. This ectoenzyme could play a protective role against the potentially deleterious effects of extracellular ATP. In tandem with 5'-nucleotidase, it produces adenosine, a powerful vasodilator, especially in hypoxic or ischemic conditions that favor the release of ATP.

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24

Alvarado-Castillo, Claudia, Patricia Lozano-Zarain, Jesús Mateo, T. Kendall Harden, and José L. Boyer. "A Fusion Protein of the Human P2Y1 Receptor and NTPDase1 Exhibits Functional Activities of the Native Receptor and Ectoenzyme and Reduced Signaling Responses to Endogenously Released Nucleotides." Molecular Pharmacology 62, no. 3 (September 1, 2002): 521–28. http://dx.doi.org/10.1124/mol.62.3.521.

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25

Belli, S. I., A. Sali, and J. W. Goding. "Divalent cations stabilize the conformation of plasma cell membrane glycoprotein PC-1 (alkaline phosphodiesterase I)." Biochemical Journal 304, no. 1 (November 15, 1994): 75–80. http://dx.doi.org/10.1042/bj3040075.

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The plasma cell-membrane glycoprotein PC-1 is an ectoenzyme with alkaline phosphodiesterase I/5′-nucleotide phosphodiesterase (EC 3.1.4.1) and nucleotide pyrophosphatase (EC 3.6.1.9) activities. It contains sequence motifs which closely match the consensus EF-hand (helix-loop-helix) Ca(2+)-binding regions of parvalbumin, troponin-C and calmodulin, and its enzymic activity is increased in the presence of divalent cations and decreased in the presence of chelating agents. We have undertaken experiments to determine whether divalent cations affect the conformation of the PC-1 protein, as assessed by their effect on thermal stability, resistance to proteolysis and binding of polyclonal antibodies to the whole native protein and monoclonal antibodies to a putative Ca(2+)-binding region. Divalent cations were found to protect solubilized PC-1 against thermal denaturation and proteolysis. They also stabilized PC-1 on intact cells; this form was much more resistant to proteolysis than Triton X-100 solubilized PC-1. Ca2+, Mg2+ and Zn2+ ions were equally effective. Monoclonal antibodies to the bacterially expressed C-terminal EF-hand homology region only bound to mammalian PC-1 in the absence of Ca2+. In contrast, the great majority of polyclonal antibodies to native PC-1 bound regardless of whether Ca2+ was present or not, but with increased binding when Ca2+ was present. These results provide evidence that divalent cations bind to PC-1 and stabilize its conformation.

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Akintunde, J. K., A. E. Irondi, E. O. Ajani, and T. V. Olayemi. "Dietary Inclusion of Black Seed Flour Inhibits Reproductive Alterations via Signal Transduction by Depleting Ectoenzyme, Adenosine Deaminase and Acetylcholine Sterase Activities in Rats Intoxicated with Mixed Environmental Metals." Journal of Biologically Active Products from Nature 8, no. 6 (November 2, 2018): 376–92. http://dx.doi.org/10.1080/22311866.2018.1523687.

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27

Casarini, D. E., M. A. Boim, R. C. Stella, M. H. Krieger-Azzolini, J. E. Krieger, and N. Schor. "Angiotensin I-converting enzyme activity in tubular fluid along the rat nephron." American Journal of Physiology-Renal Physiology 272, no. 3 (March 1, 1997): F405—F409. http://dx.doi.org/10.1152/ajprenal.1997.272.3.f405.

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The activity of angiotensin I-converting enzyme (ACE) was determined in tubular fluid collected from several portions of the rat nephron and urine and in total and efferent arteriolar blood using hippuryl-L-His-Leu as substrate. ACE activity decreased 30% from the pre- to the postglomerular arterioles (P < 0.001), suggesting a role of the glomerulus in ACE clearance. The enzyme activity was found to be present throughout the rat nephron. However, the highest activities were found in the proximal tubule and urine (0.692 +/- 0.007 and 1.05 +/- 0.015 pmol x microl(-1) x min(-1), respectively). Compared with other segments, ACE activity decreased from the initial portion of the proximal tubule to the distal nephron and increased again in the urine. Along the proximal tubule, ACE was secreted and degraded and/or reabsorbed and then secreted again into the collecting duct; no ACE activity was found in the late distal tubule, but a high level was detected in the urine, indicating a potential physiological role in the inactivation of the kinins formed by kallikrein beyond the connecting tubules. Moreover, the possible role of mesangial cells (MC) in the decrease of intraglomerular ACE was also evaluated. The analysis of ACE gene showed that MC in culture are able to express ACE mRNA. Moreover, ACE is produced as an ectoenzyme and as a secreted form of the enzyme, indicating a potential effect of local angiotensin II production on MC function.

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28

Lafrance, M. H., C. Vézina, Q. Wang, G. Boileau, P. Crine, and G. Lemay. "Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11." Biochemical Journal 302, no. 2 (September 1, 1994): 451–54. http://dx.doi.org/10.1042/bj3020451.

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Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

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29

Wong, Philip Ching Yat, Jun Guo, and Aidong Zhang. "The renal and cardiovascular effects of natriuretic peptides." Advances in Physiology Education 41, no. 2 (June 1, 2017): 179–85. http://dx.doi.org/10.1152/advan.00177.2016.

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The landmark report by de Bold et al. in 1981 signified the heart as one of the endocrine organs involved in fluid and salt balance (de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. Life Sci 28: 89–94, 1981). Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are secreted from cardiomyocytes in response to cardiac stretch as in the case of heart failure, whereas C-type natriuretic peptide (CNP) is secreted from endothelial and renal cells in response to cytokines and endothelium-dependent agonists, such as acetylcholine. Binding ANP or BNP to natriuretic peptide receptor A induces cyclic guanylyl monophosphate as second messenger in the target cells to mediate the following: natriuresis; water diuresis; increasing glomerular filtration rate; decreasing systemic sympathetic activities; plasma volume; cardiac output and blood pressure; and curbing mitoses of heart fibroblasts and hypertrophy of cardiovascular muscle cells. ANP, BNP, and CNP are cleared from the bloodstream by natriuretic peptide receptor C and degraded by an ectoenzyme called neprilysin (NEP). The plasma levels of BNP are typically >100 pg/ml in patients with congestive heart failure. Sacubitril/valsartan is an angiotensin receptor NEP inhibitor that prevents the clinical progression of surviving patients with heart failure more effectively than enalapril, an angiotensin-converting enzyme inhibitor. A thorough understanding of the renal and cardiovascular effects of natriuretic peptides is of major importance for first-year medical students to gain insight into the significance of plasma levels of BNP in patients with heart failure.

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Farhad, Hariri Akbari, Slemc Lucija, Alizadeh Mahdi, Hatami Fard Mahnaz, and Shafaei Peymaneh. "A short review of CD73 Role in Cancer." Research Journal of Biotechnology 16, no. 7 (June 25, 2021): 211–19. http://dx.doi.org/10.25303/167rjbt21121.

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The purinergic signaling pathway has been introduced as the main way in the progression of cancer and is regulated by producing a series of nucleotides. CD73 is a surface protein encoded by the NT5E gene and plays a crucial role in cell signaling. CD73 has both enzymatic and non-enzymatic functions, independent of its enzymatic activity in cells; it has been detected to be over-expressed and plays an important role in many types of cancer. However, the non-enzymatic function of CD73 has not been well studied yet. Findings from recent research show that CD73 activities are exploited as ectoenzyme to produce extracellular adenosine; in other words, there is a tumor-induced immunosuppressive mechanism for CD73. It can increase tumor proliferation and development by restricting antitumor T immunity through adenosine receptor signaling. Several factors and mechanisms have been used to control CD73 expression. Studies in various models have shown that CD73 plays a key role in the growth of tumors; it has also become an attractive therapeutic goal for scientists in many studies related to cancer. They include identifying different cancer subsets, the anticipation of drug response in patients, precipitation of purine escape pathway activity etc. Collective studies have proved that CD73 is a substantial molecule that formulates proliferation, colonialism and invasion in vitro, angiogenesis and immune escape in vivo in cancer and tumor cells. The data has shown that the potential of CD73 distinguished it as a recognizable biological marker in the next generation of treatment and research studies on cancer.

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Lazcano, Iván, Agustina Cabral, Rosa María Uribe, Lorraine Jaimes-Hoy, Mario Perello, Patricia Joseph-Bravo, Edith Sánchez-Jaramillo, and Jean-Louis Charli. "Fasting Enhances Pyroglutamyl Peptidase II Activity in Tanycytes of the Mediobasal Hypothalamus of Male Adult Rats." Endocrinology 156, no. 7 (May 5, 2015): 2713–23. http://dx.doi.org/10.1210/en.2014-1885.

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Fasting down-regulates the hypothalamus-pituitary-thyroid (HPT) axis activity through a reduction of TRH synthesis in neurons of the parvocellular paraventricular nucleus of the hypothalamus (PVN). These TRH neurons project to the median eminence (ME), where TRH terminals are close to the cytoplasmic extensions of β2 tanycytes. Tanycytes express pyroglutamyl peptidase II (PPII), the TRH-degrading ectoenzyme that controls the amount of TRH that reaches the anterior pituitary. We tested the hypothesis that regulation of ME PPII activity is another mechanism by which fasting affects the activity of the HPT axis. Semiquantitative in situ hybridization histochemistry data indicated that PPII and deiodinase 2 mRNA levels increased in tanycytes after 48 hours of fasting. This increase was transitory, followed by an increase of PPII activity in the ME, and a partial reversion of the reduction in PVN pro-TRH mRNA levels and the number of TRH neurons detected by immunohistochemistry. In fed animals, adrenalectomy and corticosterone treatment did not change ME PPII activity 72 hours later. Methimazole-induced hypothyroidism produced a profound drop in tanycytes PPII mRNA levels, which was reverted by 3 days of treatment with T4. The activity of thyroliberinase, the serum isoform of PPII, was increased at most fasting time points studied. We conclude that delayed increases in both the ME PPII as well as the thyroliberinase activities in fasted male rats may facilitate the maintenance of the deep down-regulation of the HPT axis function, despite a partial reactivation of TRH expression in the PVN.

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Pind, Steven, and Arnis Kuksis. "Further characterization of a novel phospholipase B (phospholipase A2 – lysophospholipase) from intestinal brush-border membranes." Biochemistry and Cell Biology 69, no. 5-6 (May 1, 1991): 346–57. http://dx.doi.org/10.1139/o91-054.

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The intestinal brush-border membranes of rats and guinea pigs possess a high molecular weight, calcium-independent phospholipase B (phospholipase A2 – lysophospholipase activities) with the characteristics of a digestive ectoenzyme. A combination of subcellular fractionation, Triton X-114 phase partitioning, chromatofocusing, and preparative sodium dodecyl sulphate – polyacrylamide gel electrophoresis was used to purify a full-length, although denatured, form of this enzyme from the rat. Renaturation of the gel-purified fraction confirmed that both enzyme activities were associated with this protein. Gel slices containing the purified phospholipase B were used to generate a polyclonal antiserum in rabbits that could be used for immunoblotting. The relative mobility of the phospholipase B during electrophoresis in sodium dodecyl sulphate gels was dramatically affected by the percentage of acrylamide and the presence or absence of reducing agents in the gels. This was true for both the purified protein visualized by silver-staining and following electrophoresis of the total proteins of the membrane, with the phospholipase visualized by immunoblotting. Estimates for the molecular mass of the enzyme varied from 130 to 170 kDa in 7.5% gels and from 120 to 130 kDa in 5–10% gradient gels (with a best estimate of 120 kDa). Upon solubilization from the brush-border membrane by papain digestion, the major immunoreactive band migrated with an apparent mass of 80 kDa in both the 7.5% and 5–10% gradient gels. A major cross-reactive band was detected at 97 kDa following immunoblotting of the papain-solubilized proteins from guinea pig brush-border membranes, in agreement with the size of the purified fragment reported in the literature and at 140 kDa following immunoblotting of the intact proteins. Similar immunoblotting produced reaction with a 135-kDa protein from the rabbit brush-border membrane, as well as a 95-kDa protein following papain solubilization. These results suggest that while there are species-specific apparent molecular weights, the intestinal brush-border membrane phospholipase B is conserved among species.Key words: phospholipase, brush-border membrane, intestine, phospholipid hydrolysis, antibodies.

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Murray, H., A. J. Turner, and A. J. Kenny. "The aminopeptidase activity in the human T-cell lymphoma line (Jurkat) is not at the cell surface and is not aminopeptidase N (CD-13)." Biochemical Journal 298, no. 2 (March 1, 1994): 353–60. http://dx.doi.org/10.1042/bj2980353.

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Although lymphocytes are CD-13-negative and therefore should not express the ectoenzyme aminopeptidase N (AP-N), there have been a number of reports suggesting the presence of a cell-surface aminopeptidase with many similarities to AP-N. We have determined aminopeptidase activity with 4-methyl-7-coumarylamide (NMec) derivatives of alanine, leucine, lysine and arginine in Jurkat cells (a human T-cell lymphoma line) and in HL60 cells (a CD-13-positive myeloid leukaemia line) and compared the activities with those of purified pig AP-N and human renal microvillar membranes. Jurkat cell aminopeptidase activity doubled on disrupting the cells and the sensitivity to amastatin increased. When the cells were fractionated only 4% of the activity was recovered in the membrane fraction, compared with 87% recovery for alkaline phosphatase. The profile of activities for intact Jurkat cells was Leu > Ala > Lys > Arg, changing in the cytosolic fraction to Lys > or = Arg > Leu = Ala; the profiles for intact HL60 cells and AP-N were identical, namely Ala > Leu > Arg > Lys. The Km values for the hydrolysis of Ala-NMec and Leu-NMec by Jurkat cells were 65 microM and 11 microM, in each case some 6-fold lower than those for AP-N. The pH-activity curves for the hydrolysis of Ala-NMec by Jurkat cells and human renal microvillar membranes were displaced by almost 1 pH unit and the activity was not sensitive to the anionic composition of the buffers. However, a 3-fold activation of the cytosolic activity by 0.1 M NaCl was observed with Arg-NMec as substrate. With Ala-NMec as substrate, the sensitivity of the aminopeptidase activity to inhibitors increased markedly after disrupting the cells, but still differed from that observed with purified pig AP-N; the concentrations giving 50% inhibition were as follows (values for AP-N in parentheses): amastatin. 28 nM (150 nM); bestatin, 12 microM (43 microM), probestin, 100 nM (< 10 nM), puromycin, 30 microM (> 1 mM). Anion exchange chromatography on Mono Q revealed two activities: that of peak I preferentially hydrolysed Arg-NMec, was activated by NaCl and was insensitive to amastatin; while that of peak II was strongly inhibited by amastatin and had a broad specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

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Ishihara, Katsuhiko, Ayano Yahagi, Ryo Katsumata, Akiko Shiotani, and Masanori Iseki. "Lack of bone marrow stromal cell antigen-1 (BST-1)/CD157 ameliorated colitis induced by dextran sodium sulfate (DSS)." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 17.14. http://dx.doi.org/10.4049/jimmunol.206.supp.17.14.

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Abstract Bone marrow stromal cell antigen-1(BST-1)/CD157 is GPI-anchored ectoenzyme having adenosine diphosphate(ADP)-ribosyl cyclase and cyclic ADP-ribose hydrolase activities. BST-1 also functions as a receptor for signal transduction. Recently, various groups reported novel functions of BST-1, such as regulation of intestinal or mesenchymal stem cells. Re-analyses of BST-1 deficient mice (Bst1KO) in C57BL/6 background revealed roles in defense against tuberculosis, and prevention of depressive or anxious behaviors. Thus, BST-1 is now appreciated as an entero-neuro-immune regulator. Since expression of BST-1 in the intestines is common in human and mouse, we examined the roles for BST-1 in DSS-induced colitis using Bst1KO. Drinking water containing 3% DSS was supplied ad libitum to Bst1KO and C57BL/6 (WT) in SPF condition for 5 days, and then water without DSS for 2 days. Decreases of body weights in Bst1KO were similar to WT until day 5, but since then they became significantly slower than WT. Clinical scores based on bleeding and diarrhea were similar in both genotypes. Histological analyses with HE staining revealed the ratio of destroyed epitheliums in the total colon of Bst1KO was 15% whereas that of WT was 50%. Flow cytometrical analyses revealed no difference in the cell numbers of neutrophils and macrophages increased in the colon at day 7. Quantitative real-time PCR revealed that increases of Bst1 expression in the colons of WT started on day 1 and reached to the level more than thousand times at day 5. In the colons of Bst1KO, increases of Il6 expression at day 5 were markedly decreased compared with those of WT. These data indicate that BST-1 has pro-inflammatory roles in DSS-induced colitis through positive regulation of IL-6 production.

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CAKIR-KIEFER, Céline, Hélène MULLER-STEFFNER, Norman OPPENHEIMER, and Francis SCHUBER. "Kinetic competence of the cADP-ribose–CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling." Biochemical Journal 358, no. 2 (August 24, 2001): 399–406. http://dx.doi.org/10.1042/bj3580399.

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CD38/NAD+ glycohydrolase is a type II transmembrane glycoprotein widely used to study T- and B-cell activation and differentiation. CD38 is endowed with two different activities: it is a signal transduction molecule and an ectoenzyme that converts NAD+ into ADP-ribose (NAD+ glycohydrolase activity) and small proportions of cADP-ribose (cADPR; ADP-ribosyl cyclase activity), a calcium-mobilizing metabolite, which, ultimately, can also be hydrolysed (cADPR hydrolase activity). The relationship between these two properties, and strikingly the requirement for signalling in the formation of free or enzyme-complexed cADPR, is still ill-defined. In the present study we wanted to test whether the CD38–cADPR complex is kinetically competent in the conversion of NAD+ into the reaction product ADP-ribose. In principle, such a complex could be invoked for cross-talk, via conformational changes, with neighbouring partner(s) of CD38 thus triggering the signalling phenomena. Analysis of the kinetic parameters measured for the CD38/NAD+ glycohydrolase-catalysed hydrolysis of 2′-deoxy-2′-aminoribo-NAD+ and ADP-cyclo[N1,C1′]-2′-deoxy-2′-aminoribose (slowly hydrolysable analogues of NAD+ and cADPR respectively) ruled out that the CD38–cADPR complex can accumulate under steady-state conditions. This was borne out by simulation of the prevalent kinetic mechanism of CD38, which involve the partitioning of a common E·ADP-ribosyl intermediate in the formation of the enzyme-catalysed reaction products. Using this mechanism, microscopic rate conditions were found which transform a NAD+ glycohydrolase into an ADP-ribosyl cyclase. Altogether, the present work shows that if the cross-talk with a partner depends on a conformational change of CD38, this is most probably not attributable to the formation of the CD38–cADPR complex. In line with recent results on the conformational change triggered by CD38 ligands [Berthelier, Laboureau, Boulla, Schuber and Deterre (2000) Eur. J. Biochem. 267, 3056–3064], we believe that the Michaelis CD38–NAD+ complex could play such a role instead.

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Stefanović, Nikola Z., Tatjana P. Cvetković, Radmila M. Veličković-Radovanović, Tatjana M. Jevtović-Stoimenov, Predrag M. Vlahović, Ivana R. Stojanović, and Dušica D. Pavlović. "Pharmacogenetics May Influence Tacrolimus Daily Dose, But Not Urinary Tubular Damage Markers In The Long-Term Period After Renal Transplantation." Journal of Medical Biochemistry 34, no. 4 (October 1, 2015): 422–30. http://dx.doi.org/10.1515/jomb-2015-0001.

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SummaryBackground:The primary goal of this study was to evaluate the influence of cytochrome P450 (CYP) 3A5 (6986A>G) and ABCB1 (3435C>T) polymorphisms on tacrolimus (TAC) dosage regimen and exposure. Second, we evaluated the influence of TAC dosage regimen and the tested polymorphisms on renal oxidative injury, as well as the urinary activities of tubular ectoenzymes in a long-term period after transplantation. Also, we aimed to determine the association between renal oxidative stress and tubular damage markers in the renal transplant patients.Methods:The study included 72 patients who were on TAC based immunosuppression. Allele-specific PCR was used for polymorphism determination. We measured the urinary thiobarbituric acid reactive substances (TBARS) and reactive carbonyl derivates (RCD) in order to evaluate oxidative injury, as well as the urinary activities of ectoenzymes (N-acetyl-β-D-glucosaminidase, aminopeptidase N and dipeptidyl peptidase IV) to evaluate tubular damage.Results:The carriers of CYP 3A5*1 allele required statistically higher daily doses of TAC than CYP *3/*3 carriers, as well as the carriers of C allele of ABCB1 gene compared to those with TT genotype. Also, there were no differences in TBARS, RCD and the activities of ectoenzymes between the patients’ genotypes. Our results showed significant correlations between urinary TBARS and RCD and the ectoenzymes’ activities.Conclusions:Our findings suggest that CYP 3A5 and ABCB1 3435 polymorphism may affect TAC daily doses, but not the drug’s tubular toxicity. Furthermore, tubular damage may be associated with increased renal oxidative stress.

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Cvetković, Tatjana, Predrag Vlahović, Vidosava đorđević, Lilika Zvezdanović, Dušica Pavlović, Gordana Kocić, and Dušan Sokolović. "The Significance of Urinary Markers in the Evaluation of Diabetic Nephropathy." Journal of Medical Biochemistry 27, no. 3 (July 1, 2008): 376–82. http://dx.doi.org/10.2478/v10011-008-0019-y.

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The Significance of Urinary Markers in the Evaluation of Diabetic Nephropathy Oxidative stress is considered to be a unifying link between diabetes mellitus (DM) and its complications, including nephropathy (DN). The aim of this study was to determine the parameters of oxidative injury of lipids and proteins as well as the activity of ectoenzymes in the urine of DN patients. The study included 40 individuals: 10 patients with type 2 diabetes mellitus and microalbuminuria (DMT2-MIA), 10 type 2 diabetic patients with macroalbuminuria (DMT2-MAA), 10 patients with type 1 diabetes and microalbuminuria (DMT1-MIA) and 10 age- and sex-matched healthy subjects (control). In the urine we determined TBA reactive substances (TBARS), reactive carbonyl groups (RCG), and the activity of ectoenzymes N-acetyl-β-d-glucosaminidase (NAG), plasma cell differentiation antigen (PC-1), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPP IV). A higher concentration of TBARS in the urine was found in DMT2-MIA and DMT1-MIA, compared to the control group (p<0.001 and P<0.05). The urine concentration of RCD shows similar results with a significant elevation in the groups with DMT2-MAA and DMT1-MIA, compared to the DMT2-MIA (p<0.001) and control group (p<0.001). Activities of NAG, APN and DPPIV were significantly higher in the urine of DMT2-MAA, compared to the control (p<0.01). The activity of PC-1 was slightly increased in that group, but not significantly. In conclusion, the level of oxidative stress markers and activities of brush border ectoenzymes in the urine may be a useful non-invasive and easily repeatable test in DN.

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Abdalla, Fátima Husein, Andréia Machado Cardoso, Luciane Belmonte Pereira, Roberta Schmatz, Jamile Fabbrin Gonçalves, Naiara Stefanello, Amanda Maino Fiorenza, et al. "Neuroprotective effect of quercetin in ectoenzymes and acetylcholinesterase activities in cerebral cortex synaptosomes of cadmium-exposed rats." Molecular and Cellular Biochemistry 381, no. 1-2 (June 25, 2013): 1–8. http://dx.doi.org/10.1007/s11010-013-1659-x.

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Morandi, F., A. L. Horenstein, R. Rizzo, and F. Malavasi. "The Role of Extracellular Adenosine Generation in the Development of Autoimmune Diseases." Mediators of Inflammation 2018 (2018): 1–10. http://dx.doi.org/10.1155/2018/7019398.

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Adenosine (ADO) is an immunosuppressive molecule, which suppresses the immune responses by interacting with specific receptors expressed by immune effector cells. ADO is produced from ATP through the enzymatic activities of CD39 and CD73. Alternatively, ADO can be generated starting from NAD+, which is metabolized by the concerted action of CD38, CD203a/PC-1, and CD73. The role of ADO in immunity has been characterized in the last years in physiology and in pathological settings. This review examines a panel of reports focused on the functions of ADO in the context of human autoimmune/inflammatory diseases and the selected animal models. The final aim is to consider the role of adenosinergic ectoenzymes and ADO receptors as novel therapeutic targets for selected diseases.

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YEGUTKIN, Gennady G., Tiina HENTTINEN, Sergei S. SAMBURSKI, Jozef SPYCHALA, and Sirpa JALKANEN. "The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells." Biochemical Journal 367, no. 1 (October 1, 2002): 121–28. http://dx.doi.org/10.1042/bj20020439.

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Extracellular purines are important signalling molecules in the vasculature that are regulated by a network of cell surface ectoenzymes. By using human endothelial cells and normal and leukaemic lymphocytes as enzyme sources, we identified the following purine-converting ectoenzymes: (1) ecto-nucleotidases, NTP diphosphohydrolase/CD39 (EC 3.6.1.5) and ecto-5′-nucleotidase/CD73 (EC 3.1.3.5); (2) ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and nucleoside diphosphate kinase (EC 2.7.4.6); (3) ecto-adenosine deaminase (EC 3.5.4.4). Evidence for this was obtained by using enzyme assays with 3H-labelled nucleotides and adenosine as substrates, direct evaluation of γ-phosphate transfer from [γ-32P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20kDa surface protein was observed following incubation of Namalwa B cells with [γ-32P]ATP. Thus two opposite, ATP-generating and ATP-consuming, pathways coexist on the cell surface, where basal ATP release, re-synthesis of high-energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent adenosine inactivation. The comparative measurements of enzymic activities indicated the predominance of the nucleotide-inactivating pathway via ecto-nucleotidase reactions on the endothelial cells. The lymphocytes are characterized by counteracting ATP-regenerating/adenosine-eliminating phenotypes, thus allowing them to avoid the lymphotoxic effects of adenosine and maintain surrounding ATP at a steady-state level. These results are in agreement with divergent effects of ATP and adenosine on endothelial function and haemostasis, and provide a novel regulatory mechanism of local agonist availability for nucleotide- or nucleoside-selective receptors within the vasculature.

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Soares, Mayara Sandrielly Pereira, Marcelo Zanusso Costa, Tatiane Morgana da Silva, Marta Gazal, Carlus Augustu Tavares do Couto, Gabriela Nogueira Debom, Rodrigo Rodrigues, et al. "Methionine and/or Methionine Sulfoxide Alter Ectoenzymes Activities in Lymphocytes and Inflammatory Parameters in Serum from Young Rats: Acute and Chronic Effects." Cell Biochemistry and Biophysics 76, no. 1-2 (July 19, 2017): 243–53. http://dx.doi.org/10.1007/s12013-017-0815-4.

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Delgado, Jerónimo, Ana Saborido, María Morán, and Alicia Megías. "Chronic and acute exercise do not alter Ca2+ regulatory systems and ectonucleotidase activities in rat heart." Journal of Applied Physiology 87, no. 1 (July 1, 1999): 152–60. http://dx.doi.org/10.1152/jappl.1999.87.1.152.

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The purpose of this investigation was to examine the effects of chronic and acute exercise on the main components involved in excitation-contraction coupling and relaxation in rat heart. Sixty male Wistar rats were divided into a sedentary (S) and three 12-wk treadmill-trained groups (T-1, moderate intensity; T-2, high intensity; T-3, interval running). After 12-wk, 15 rats from the S group and 15 rats from the T-2 group were subjected to a single treadmill-exercise session until exhaustion before being killed at 0, 24, or 48 h (acute exercise). The remaining animals were killed 48 h after the last standard exercise session (chronic exercise). The efficacy of the training programs was confirmed by an increase in treadmill endurance time and in skeletal muscle citrate synthase activity. None of the exercise programs modified heart weight or cardiac oxidative capacity. [3H]PN200–110 and [3H]ryanodine binding to cardiac homogenates indicated that the density of L-type and sarcoplasmic reticulum (SR) Ca2+channels was the same in S and trained rats. The SR Ca2+-ATPase activity was also unmodified. Finally, the activities of the ectoenzymes Mg2+-ATPase and 5′-nucleotidase, which are involved in degradation of extracellular nucleotides, were not affected by either of the running programs. After the acute exercise session, no changes were detected in either of the tested parameters in heart homogenates of S and T-2 animals. We conclude that neither treadmill-exercise training for 12 wk nor exhaustive exercise alters the density of Ca2+ channels involved in excitation-contraction coupling or the SR Ca2+-ATPase and the ectonucleotidase activities in rat heart.

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Iijima, Osamu, Koichi Miyake, Aki Nakamura, Tsutomu Igarashi, Chizu Kanokoda, Atsushi Watanabe, and Takashi Shimada. "Bone Marrow Cell Based Enzyme Replacement Prolongs Survival and Improves Disease Phenotypes In a Mouse Model Of Lethal Hypophosphatasia." Blood 122, no. 21 (November 15, 2013): 2897. http://dx.doi.org/10.1182/blood.v122.21.2897.2897.

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Abstract Hypophosphatasia (HPP) is an inherited skeletal disease caused by mutations of the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNALP). TNALP is an ectoenzyme and plays an essential role in bone mineralization. The major symptoms of HPP are hypomineralization of systemic bones, respiratory insufficiency and epileptic seizures. Perinatal and infantile forms of HPP are often fatal. Since ALP functions on the exterior of the cells, enzyme replacement therapy (ERT) is a potential approach to treat HPP. Currently, Phase II/III clinical trials of ERT using a recombinant TNALP which linked deca-aspartate (D10) at the C terminus for bone targeting are ongoing in North America, Europe and Japan. The perinatal and infantile patients received the ERT showed apparent improvement of the symptoms. However, the ERT is highly invasive for the young patients because it requires repeated subcutaneous administration of large amounts of the enzyme every 3 times a week for long-term correction. As another approach to treat HPP, we have reported in vivo gene therapy for ALPL (Akp2) knock-out mice (HPP mice). The treated HPP mice were rescued by a single systemic injection of lentiviral vector or adeno-associated viral vector expressing bone targeted form of TNALP (TNALP-D10) during the neonatal or fetal period. Although untreated HPP mice developed apparent growth failure and died by around 20 days of age due to severe skeletal hypomineralization and epileptic seizure, the treated HPP mice were prolonged the survival and improved the physical activity. In the treated HPP mice, plasma ALP activity was kept higher than 1 U/ml (approximately 0.01 U/ml in untreated HPP mice and 0.1 U/ml in wild type (WT) mice) which gives therapeutic effects. However, disadvantages of in vivo gene therapy include the risk of germline gene transfer and induction of immune responses to the vectors or transgene products. To overcome these problems, we examined a feasibility of ex vivo gene therapy using hematopoietic stem cells (HSC) transduced by lentiviral vector expressing TNALP-D10. The potential advantages of this approach are lifelong expression of TNALP-D10 and prevention of risks of in vivo gene therapy. The lineage negative bone marrow cells containing HSC (Lin- BMC) were harvested from B6.CD45.1 mice (Ly5.1) and then enriched using Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set (BD bioscience). Lin- BMC was transduced with lentiviral vector expressing TNALP-D10 for 20 hrs at an moi of 50 with mSCF, mIL3 and rhIL6 on Retronectin coated plate. Recipient HPP mice (Ly5.2) on day 2 after birth were received a sub-lethal dose of total body irradiation (4Gy) 4hr prior to transplantation. Then, the transduced Lin- BMC (1 x 106 cells) was transplanted intravenously into the HPP mice through the temporal vein or jugular vein. The plasma ALP activity was rapidly elevated approximately 400 fold higher than untreated HPP mice (untreated: 0.014±0.004 units/ml (n=4) and treated: 5.39±2.29 units/ml (n=7), respectively) on 1 week after the transplantation and kept at this level during the observation period. Engraftment rate of Ly5.1 donor cells were sustained at approximately 30-40% with multilineage potential. The treated HPP mice were prolonged their survival over 3 months without epileptic seizures and the physical activities were improved. The histochemical ALP staining indicated TNALP-D10 was accumulated on the surface of trabecular and cortical bones of the treated HPP mice. The bone mineralization was significantly improved, but still not satisfactory compared with age matched WT mice. Contrary to our expectations, 2 of 9 HPP mice transplanted with non-transduced BMC also survived for 3 months. However, the plasma ALP activity was not elevated at all and the bone mineralization was incomplete compared with treated HPP mice. These results indicate that a single transplantation of genetically modified BMC at neonatal period is sufficient for long-term supply of TNALP-D10 and rescue of lethal HPP mice, even though hypomineralization was not completely recovered. Further optimization of viral vector and conditioning of transplantation is required to increase the treatment efficacy for HPP. However, neonatal ex vivo gene therapy using genetically modified BMC would be a possible and practical approach to treat HPP. Disclosures: Watanabe: Alexion Pharmaceuticals, Inc.: Membership on an entity’s Board of Directors or advisory committees.

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Sindhu, K. J., Amit Kumar Kureel, Sheetal Saini, Smita Kumari, Pankaj Verma, and Ambak Kumar Rai. "Characterization of phosphate transporter(s) and understanding their role in Leishmania donovani parasite." Acta Parasitologica 63, no. 1 (March 26, 2018): 75–88. http://dx.doi.org/10.1515/ap-2018-0009.

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Abstract Inorganic phosphate (Pi) is shown to be involved in excretion of methylglyoxal (MG) in the promastigote form of Leishmania donovani parasite. Absence of Pi leads to its accumulation inside the parasite. Accumulation of MG is toxic to the parasite and utilizes glyoxylase as well as excretory pathways for its detoxification. In addition, Pi is also reported to regulate activities of ectoenzymes and energy metabolism (glucose to pyruvate) etc. Thus, it is known to cumulatively affect the growth of Leishmania parasite. Hence the transporters, which allow the movement of Pi across the membrane, can prove to be a crucial drug target. Therefore, we characterized two phosphate transporters in Leishmania (i) H+ dependent myo-inositol transporter (LdPHO84), and (ii) Na+ dependent transporter (LdPHO89), based on similar studies done previously on other lower organisms and trypanosomatids. We tried to understand the secondary structure of these two proteins and confirm modulation in their expression with the change in Pi concentration outside. Moreover, their modes of action were also measured in the presence of specific inhibitors (LiF, CCCP). Further analysis on the physiological role of these transporters in various stages of the parasite life cycle needs to be entrenched.

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Franco, Luisa, Santina Bruzzone, Pinfang Song, Lucrezia Guida, Elena Zocchi, Timothy F. Walseth, Emanuele Crimi, Cesare Usai, Antonio De Flora, and Vito Brusasco. "Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle." American Journal of Physiology-Lung Cellular and Molecular Physiology 280, no. 1 (January 1, 2001): L98—L106. http://dx.doi.org/10.1152/ajplung.2001.280.1.l98.

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Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD+ by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 μM) with an increase in intracellular calcium concentration ([Ca2+]i) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH2-cADPR (10 μM) inhibited both basal and ACh-stimulated [Ca2+]i levels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH2-cADPR decreased it. Tracheal mucosa strips, by releasing NAD+, enhanced [Ca2+]iin isolated TSMs, and this increase was abrogated by either NAD+-ase or 8-NH2-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD+ plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.

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Gasparrini, Massimiliano, Leonardo Sorci, and Nadia Raffaelli. "Enzymology of extracellular NAD metabolism." Cellular and Molecular Life Sciences 78, no. 7 (March 23, 2021): 3317–31. http://dx.doi.org/10.1007/s00018-020-03742-1.

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AbstractExtracellular NAD represents a key signaling molecule in different physiological and pathological conditions. It exerts such function both directly, through the activation of specific purinergic receptors, or indirectly, serving as substrate of ectoenzymes, such as CD73, nucleotide pyrophosphatase/phosphodiesterase 1, CD38 and its paralog CD157, and ecto ADP ribosyltransferases. By hydrolyzing NAD, these enzymes dictate extracellular NAD availability, thus regulating its direct signaling role. In addition, they can generate from NAD smaller signaling molecules, like the immunomodulator adenosine, or they can use NAD to ADP-ribosylate various extracellular proteins and membrane receptors, with significant impact on the control of immunity, inflammatory response, tumorigenesis, and other diseases. Besides, they release from NAD several pyridine metabolites that can be taken up by the cell for the intracellular regeneration of NAD itself. The extracellular environment also hosts nicotinamide phosphoribosyltransferase and nicotinic acid phosphoribosyltransferase, which inside the cell catalyze key reactions in NAD salvaging pathways. The extracellular forms of these enzymes behave as cytokines, with pro-inflammatory functions. This review summarizes the current knowledge on the extracellular NAD metabolome and describes the major biochemical properties of the enzymes involved in extracellular NAD metabolism, focusing on the contribution of their catalytic activities to the biological function. By uncovering the controversies and gaps in their characterization, further research directions are suggested, also to better exploit the great potential of these enzymes as therapeutic targets in various human diseases.

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Shayhidin, Elnur E., Elsa Forcellini, Marie-Chloé Boulanger, Mahmut Ablajan, Sébastien Dautrey, Xavier Barbeau, Patrick Lague, Jean Sévigny, Jean-François Paquin, and Patrick Mathieu. "Abstract 16349: A Novel Class of Inhibitors That Are Specific to Human NPP1 Prevent Pathologic Mineralization of Valve Interstitial Cells." Circulation 130, suppl_2 (November 25, 2014). http://dx.doi.org/10.1161/circ.130.suppl_2.16349.

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BACKGROUND: Ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an ectoenzyme, which plays a role into several disorders including calcific aortic valve disease (CAVD). So far, compounds that have been developed as inhibitors of NPP1 lack of potency and specificity. Quinazolin-4-piperidin-4-methyl sulfamide (QPS) derivatives have been described as potent inhibitors of NPP1. However, their mode of inhibition as well as their selectivity and capacity to modify biological processes have not been investigated. METHOD: We have investigated the potency and selectivity of QPS derivatives in inhibiting NPP1 by enzymatic activities. The biological effect of QPS derivatives was documented on the mineralization of valve interstitial cell cultures, apoptosis assay, as well as quantitative polymerase chain reaction. RESULTS: We documented that QPS1 derivative is a potent (67.9 ±5.3 nM) and selective non-competitive inhibitor of human NPP1. Moreover, QPS1 also significantly inhibited the K121Q NPP1 gene variant (ki 51.9±9.8 nM), which is prevalent in the general population. QPS1 did not significantly alter the activity of other nucleotide metabolising enzymes expressed at the cell surface, namely NPP3, NTPDases (1-3), ecto-5’-nucleotidase and ALP. Importantly, QPS1 in the low micromolar range (≤10μM) prevented phosphate-induced mineralization of VICs and lowered the rise of osteogenic genes as expected for NPP1 inhibition. CONCLUSION: We provide evidence that QPS1 is a potent and selective non-competitive inhibitor of NPP1 that prevents pathologic mineralization in a cellular model.

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Adefegha, Stephen Adeniyi, Renata da Silva Pereira Saccol, Matheus Henrique Jantsch, Karine Lanes da Silveira, and Daniela Bitencourt Rosa Leal. "Hesperidin mitigates inflammation and modulates ectoenzymes activity and some cellular processes in complete Freund’s adjuvant-induced arthritic rats." Journal of Pharmacy and Pharmacology, August 24, 2021. http://dx.doi.org/10.1093/jpp/rgab100.

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Abstract Objectives This study was aimed at assessing the anti-arthritic effects of hesperidin on the inflammatory markers in serum/plasma, ectoenzymes activity in platelet, reactive oxygen species (ROS), apoptosis and cell cycle in bone marrow cells of a rat model of arthritis. Methods Fifty-six adult female Wistar rats (245–274 g) were grouped into eight of seven rats each: control rats given normal saline or 40 mg/kg of hesperidin or 80 mg/kg of hesperidin, 0.2 mg/kg of dexamethasone, arthritic rats given normal saline, or 40 mg/kg of hesperidin or 80 mg/kg of hesperidin, and 0.2 mg/kg of dexamethasone. Myeloperoxidase and nitrate plus nitrite levels were evaluated in the plasma and serum, respectively. The ecto-nucleoside triphosphate diphosphohydrolases, ecto-5′-nucleotidase and ecto-adenosine deaminase activities were assessed in platelets. Subsequently, the cells of the bone marrow were obtained, and the assays for ROS, apoptosis and cell cycle were evaluated using flow cytometry. Key findings The results showed that hesperidin mitigated inflammation, modulated adenosine nucleotides and nucleoside hydrolysing enzymes and levels, minimized ROS intracellularly, attenuated apoptotic process and activated cell cycle arrest in arthritic rat. Conclusion This study suggests that hesperidin could be a natural and promising anti-inflammatory compound for the management of arthritis.

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Fraser-Pitt, Douglas J., Derry K. Mercer, Daniel Smith, Aleksandra Kowalczuk, Jennifer Robertson, Emma Lovie, Peter Perenyi, et al. "Cysteamine, an Endogenous Aminothiol, and Cystamine, the Disulfide Product of Oxidation, IncreasePseudomonas aeruginosaSensitivity to Reactive Oxygen and Nitrogen Species and Potentiate Therapeutic Antibiotics against Bacterial Infection." Infection and Immunity 86, no. 6 (March 26, 2018). http://dx.doi.org/10.1128/iai.00947-17.

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ABSTRACTCysteamine is an endogenous aminothiol produced in mammalian cells as a consequence of coenzyme A metabolism through the activity of the vanin family of pantetheinase ectoenzymes. It is known to have a biological role in oxidative stress, inflammation, and cell migration. There have been several reports demonstrating anti-infective properties targeting viruses, bacteria, and even the malarial parasite. We and others have previously described broad-spectrum antimicrobial and antibiofilm activities of cysteamine. Here, we go further to demonstrate redox-dependent mechanisms of action for the compound and how its antimicrobial effects are, at least in part, due to undermining bacterial defenses against oxidative and nitrosative challenges. We demonstrate the therapeutic potentiation of antibiotic therapy againstPseudomonas aeruginosain mouse models of infection. We also demonstrate potentiation of many different classes of antibiotics against a selection of priority antibiotic-resistant pathogens, including colistin (often considered an antibiotic of last resort), and we discuss how this endogenous antimicrobial component of innate immunity has a role in infectious disease that is beginning to be explored and is not yet fully understood.

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"Leaves and seeds of Syzygium cumini extracts produce significant attenuation of 2,2 azobis-2-amidinopropane dihydrochloride-induced toxicity via modulation of ectoenzymes and antioxidant activities." Journal of Applied Pharmaceutical Science, 2017. http://dx.doi.org/10.7324/japs.2017.70606.

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Journal articles on the topic 'Ectoenzyme activities'

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