Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated changes in angiotensin converting-enzyme (ACE) activity before and at 5, 15, 60, and 240 min after 20 micrograms phorbol myristate acetate/kg body wt iv in conscious rabbits. ACE activity was estimated in vivo from the single-pass transpulmonary metabolism of the synthetic substrate [3H]benzoyl-Phe-Ala-Pro [( 3H]BPAP) under first-order reaction conditions. Within 5 min after PMA administration, all animals developed profound granulocytopenia (15% of control) and moderate thrombocytopenia (57% of control), both lasting for the duration of the experiment. Concomitantly, there was a significant decrease in the transpulmonary metabolism of [3H]BPAP and the calculated apparent first-order reaction constant Amax/Km of ACE for [3H]BPAP. No histological evidence of lung injury was observed at these times. Since a concomitant fall in the permeability surface area product for urea was also observed, we considered that the apparent decline in ACE activity might have resulted from a reduction in perfused endothelial surface area. To resolve this, we studied the effect of PMA on the Km (a measure of enzyme affinity for its substrate) and Amax (a derivative of Vmax that is dependent upon total enzyme present and thus capillary surface area) of ACE and 5'-nucleotidase for [3H]BPAP and [14C]AMP, respectively. A significant increase in Km for both enzymes was observed at 1 h after PMA, whereas Amax was unaffected, suggesting that low-dose PMA may indeed produce endothelial cell enzyme dysfunction independent of its effect on capillary surface area. These results provide evidence of pulmonary capillary functional injury before or in the absence of structural endothelial damage.
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PMID:Early pulmonary endothelial enzyme dysfunction after phorbol ester in conscious rabbits. 369 30

Angiotensin-converting enzyme and 5'-nucleotidase line the luminal surface of pulmonary microvascular endothelium and participate in the synthesis and/or degradation of potent vasoactive substances. We applied Michaelis-Menten kinetics in simultaneous estimations of apparent constants Km and Amax (product of Vmax and microvascular plasma volume) of these two enzymes for the substrates 3H-labeled benzoyl-Phe-Ala-Pro and 14C-labeled 5'-AMP, respectively, in vivo. Values of angiotensin-converting enzyme for benzoyl-Phe-Ala-Pro (Km = 10-11 microM; Amax = 12-13 mumol X min-1) were somewhat higher than published estimates in vitro and changed predictably in response to the known enzyme inhibitor captopril. Kinetic values of 5'-nucleotidase for 5'-AMP (Km = 3-4 microM; Amax = 3-4 mumol/min) were substantially lower than those reported in vitro but also responded predictably to the competitive inhibitor of 5'-nucleotidase, adenosine 5'-[alpha, beta-methylene]diphosphate. These data offer in vivo estimates of enzyme kinetics that are useful in revealing enzyme behavior in their normal physiological environment and provide means of evaluating the action of pharmacological, physiological, and pathological modulators of enzyme activity, in vivo.
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PMID:Kinetics of pulmonary angiotensin-converting enzyme and 5'-nucleotidase in vivo. 609 4

Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.
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PMID:Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C. 798 Apr 26

We investigated the early effects of radiation on pulmonary endothelial function in vivo 7-8 hr after exposure of rabbits to a single dose of 30 Gy to the chest. Utilizing multiple indicator-dilution techniques, we measured rates and kinetics of hydrolysis of the synthetic substrates [3H]benzoyl-Phe-Ala-Pro (BPAP) and [14C]benzoyl-Ala-Gly-Pro (BAGP) by endothelial-bound angiotensin-converting enzyme (ACE) and of 5'[14C]-AMP by endothelial-bound 5'-nucleotidase (NCT) and binding of the synthetic ACE inhibitor [3H]RAC-X-65 during a single transpulmonary passage in anesthetized, artificially ventilated, open-chest rabbits in which both systemic and pulmonary circulations were fully supported by an extracorporeal pump. We have shown that these techniques and the use of the aforementioned probes provide reliable information on pulmonary endothelial function in vivo. Radiation to the chest produced endothelial ectoenzyme dysfunction, as reflected in altered available perfused capillary surface area and altered enzyme kinetics of all probes (decreases in substrate hydrolysis, inhibitor binding, first- and second-order kinetic constants) over a wide range of pulmonary blood flow values (reflecting approximately 60-200% of normal cardiac output). Indomethacin prevented most of these alterations in partially as well as fully recruited lungs. We conclude that impairment of endothelial ectoenzyme activity is an early event in the pathogenesis of radiation-induced lung damage, which occurs independently of hemodynamic influences and may involve synthesis of arachidonic acid metabolites.
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PMID:Radiation-induced early pulmonary endothelial ectoenzyme dysfunction in vivo: effect of indomethacin. 829 Oct 52

We monitored the activity of pulmonary microvascular endothelial-bound angiotensin-converting enzyme (ACE) in vivo by means of multiple indicator-dilution-type techniques, utilizing three different probes: the hydrolysis of two substrates, [3H]-benzoyl-Phe-Ala-Pro (BPAP) and [14C]benzoyl-Ala-Gly-Pro (BAGP), and the binding of the inhibitor [3H]RAC-X-65 (RAC), all measured during a single transpulmonary passage in anesthetized rabbits, placed on total heart bypass, so that both systemic and pulmonary circulations were fully supported by means of a two-channel extracorporeal pump. Experiments were performed at pulmonary blood flows (Qb) of 250, 400, 560, and 800 ml/min in control or indomethacin-pretreated rabbits. ACE activity was also compared to that of pulmonary microvascular endothelial-bound 5'-nucleotidase, by measuring the dephosphorylation of its natural substrate 5'-[14C]AMP. We calculated substrate utilization, mean lung transit time (t), and volume of distribution (i.e., central blood volume) of all substrates, as well as inhibitor binding. We also calculated Amax/Km and Bmax products of enzyme mass and kinetic constants for substrates and inhibitor, respectively. As Qb increased, Amax/Km values for all three substrates and Bmax increased linearly, indicating microvascular recruitment. In experiments in which either BPAP and 5'-AMP metabolism or BAGP metabolism and RAC binding were studied concomitantly, a linear relationship was observed between Qb-induced changes in Amax/Km values of BPAP vs 5'-AMP as well as in Amax/Km of BAGP vs Bmax of RAC. Similarly, increasing Qb increased central blood volume and decreased t. Indomethacin had no effect on most of the hemodynamic or enzyme parameters measured. We conclude that in vivo assays of ACE proceed as predicted by Michaelis-Menten kinetics and offer insights into pulmonary endothelial pathophysiology.
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PMID:Assay of pulmonary microvascular endothelial angiotensin-converting enzyme in vivo: comparison of three probes. 829 Oct 66

To examine whether activation of polymorphonuclear leukocytes attenuates release of adenosine through attenuation of their own ecto-5'-nucleotidase activity, human polymorphonuclear leukocytes were incubated with and without exposure to either N-formyl-methionyl-leucyl-phenylalanine (FMLP) or complement C5a. Ecto-5'-nucleotidase activity of polymorphonuclear leukocytes was attenuated by both FMLP and complement C5a (22.7 +/- 3.6 vs 9.7 +/- 2.6 nmol/min per 10(7) cells at 10(-6) M FMLP, P < .05; 21.5 +/- 2.2 vs 10.2 +/- 1.2 nmol/min per 10(7) cells at 5 x 10(-7) g/mL complement C5a, P < .001), whereas cytosolic 5'-nucleotidase activity was not affected by either FMLP or complement C5a. These reductions of ecto-5'-nucleotidase activity that were caused by both FMLP and complement C5a were dose and time dependent and were inhibited by superoxide dismutase. Desferrioxamine did not inhibit the decreases in ecto-5'-nucleotidase. In accordance with the decreases in ecto-5'-nucleotidase activity, release of adenosine was attenuated in the FMLP-pretreated and complement C5a-pretreated polymorphonuclear leukocytes, which were restored by concomitant administration of superoxide dismutase. The viability of FMLP-pretreated and complement C5a-pretreated polymorphonuclear leukocytes was markedly decreased compared with the untreated group after 60 minutes of hypoxia followed by 60 minutes of reoxygenation. Thus, we conclude that: (1) activation of polymorphonuclear leukocytes attenuates their own ecto-5'-nucleotidase activity and thereby reduces adenosine release, (2) reduction of ecto-5'-nucleotidase activity is attributable to generated superoxide anion in polymorphonuclear leukocytes, and (3) viability of polymorphonuclear leukocytes after hypoxia and reoxygenation largely depends on the extents of decreases in ecto-5'-nucleotidase activity.
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PMID:Attenuation of ecto-5'-nucleotidase activity and adenosine release in activated human polymorphonuclear leukocytes. 834 95

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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PMID:Relationship between volume of bathing medium and ectoenzyme activity in monolayers of cultured BPAEC. 884 96

We investigated pulmonary endothelial function in vivo in 12- to 18-mo-old male Watanabe heritable hyperlipidemic (WHHL; n = 7) and age- and sex-matched New Zealand White (n = 8) rabbits. The animals were anesthetized and artificially ventilated, and the chest was opened and put in total heart bypass. The single-pass transpulmonary utilizations of the angiotensin-converting enzyme (ACE) substrate [(3)H]benzoyl-Phe-Ala-Pro (BPAP) and the 5'-nucleotidase (NCT) substrate [(14)C]AMP were estimated, and the first-order reaction parameter A(max)/K(m), where A(max) is the product of enzyme mass and the catalytic rate constant and K(m) is the Michaelis-Menten constant, was calculated. BPAP transpulmonary utilization and A(max)/K(m) were reduced in WHHL (1.69 +/- 0.16 vs. 2.9 +/- 0.44 and 599 +/- 69 vs. 987 +/- 153 ml/min in WHHL and control rabbits, respectively; P < 0.05 for both). No differences were observed in the AMP parameters. BPAP K(m) and A(max) values were estimated separately under mixed-order reaction conditions. No differences in K(m) values were found (9.79 +/- 1 vs. 9.9 +/- 1.31microM), whereas WHHL rabbit A(max) was significantly decreased (5.29 +/- 0.88 vs. 7. 93 +/- 0.8 micromol/min in WHHL and control rabbits, respectively; P < 0.05). We conclude that the observed pulmonary endothelial ACE activity reduction in WHHL rabbits appears related to a decrease in enzyme mass rather than to alterations in enzyme affinity.
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PMID:Reduced lung endothelial angiotensin-converting enzyme activity in Watanabe hyperlipidemic rabbits in vivo. 1083 35

The main objective of the present study was to characterize the inhibition by phenylalanine and phenylpyruvate of ATP diphosphohydrolase activity in synaptosomes from the brain cortex of rats. This enzyme participates together with a 5'-nucleotidase in adenosine formation from the neurotransmitter, ATP, in the synaptic cleft. The inhibition of ATP diphosphohydrolase was competitive for nucleotide hydrolysis but 5'-nucleotidase was not affected by these metabolites. Furthermore, the two substances inhibited enzyme activity by acting at the same binding site. If the enzyme inhibition observed in vitro also occurs in the brain of PKU patients, it may promote an increase in ATP levels in the synaptic cleft. In this case, the neurotoxicity of ATP could possibly be one of the mechanisms leading to the characteristic brain damage of phenylketonuria.
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PMID:Phenylalanine and phenylpyruvate inhibit ATP diphosphohydrolase from rat brain cortex. 1170 69

Cytosolic 5'-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5'-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5'-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues.
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PMID:Structural insights into the inhibition of cytosolic 5'-nucleotidase II (cN-II) by ribonucleoside 5'-monophosphate analogues. 2217 67


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