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PubMed1987

Xanthine oxidase is not responsible for reoxygenation injury in isolated-perfused rat heart.

The massive leakage of intracellular enzymes which occurs during reoxygenation of heart tissue after hypoxic or ischemic episodes has been suggested to result from the formation of oxygen radicals. One purported source of such radicals is the xanthine oxidase-mediated metabolism of hypoxanthine and xanthine. Xanthine oxidase (O form) has been suggested to be formed in vivo by limited proteolysis of xanthine dehydrogenase (D form) during the hypoxic period (Granger et al., Gastroenterology, 81, 22 (1981)). We measured the activities of xanthine oxidase in both fresh and isolated-perfused (Langendorff) rat heart tissue. Approximately 32% of the total xanthine oxidase was in the O form in fresh and isolated-perfused rat heart. This value was unchanged following 60 min of hypoxia and 30 minutes of reoxygenation. The infusion of 250 microM allopurinol throughout the perfusion completely inhibited xanthine oxidase activity but had no effect on the massive release of lactate dehydrogenase (LDH) into the coronary effluent upon reoxygenation of heart tissue subjected to 30 or 60 min of hypoxia. Protection from 30 min of hypoxia was also not obtained when rats were pretreated for 48 h with allopurinol at a dose of 30 mg/kg/day and perfused with allopurinol containing medium. Superoxide dismutase (50 units/ml), catalase (200 units/ml), or the antioxidant cyanidanol (100 microM) also had no effect on LDH release upon reoxygenation after 60 min of hypoxia. Xanthine oxidase activity was detected in a preparation enriched in cardiac endothelial cells while no allopurinol-inhibitable activity could be measured in purified isolated cardiomyocytes. It is concluded that xanthine dehydrogenase is not converted to xanthine oxidase in hypoxic tissue of the isolated perfused rat heart, and that the release of intracellular enzymes upon reoxygenation in this experimental model is mediated by factors other than reactive oxygen generated by xanthine oxidase.

Allopurinol↗
PubMed1986

Polyamine oxidase in human retroplacental serum inhibits the growth of Plasmodium falciparum.

Human retroplacental serum (RPS) containing polyamine oxidase inhibited the growth of the Camp strain of Plasmodium falciparum in vitro as assayed by the parasite's decreased incorporation of 3H-hypoxanthine. Inhibition was dose-dependent on the concentrations of serum polyamine oxidase and added polyamines. Almost complete inhibition was seen in 96-hr asynchronous cultures containing 10% RPS and in those containing 1.2% RPS plus 50 microM polyamine. Subtle morphologic changes in mature stages and decreased numbers of new rings were associated with inhibition seen in 19-hr synchronous cultures initiated at the trophozoite stage. These incubation times were longer than in previous reports showing inhibition of malaria parasites by bovine polyamine oxidase but not by human polyamine oxidase. Macrophages contain polyamine oxidase, the reaction products of which are known to be similar to those of RPS polyamine oxidase but different from those of bovine polyamine oxidase. It remains to be determined whether human polyamine oxidase, acting upon ubiquitous polyamines, contributes to host defenses against malaria.

Animals↗
PubMed2006

Tat-mediated protein transduction of human brain pyridoxine-5-P oxidase into PC12 cells.

Pyridoxine-5-P oxidase catalyses the terminal step in the biosynthesis of pyridoxal-5-P, the biologically active form of vitamin B6 which acts as an essential cofactor. Here, a human brain pyridoxine-5-P oxidase gene was fused with a gene fragment encoding the HIV-1 Tat protein transduction domain (RKKRRQRRR) in a bacterial expression vector to produce a genetic in-frame Tat-pyridoxine-5-P oxidase fusion protein. Expressed and purified Tat-pyridoxine-5-P oxidase fusion protein transduced efficiently into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced Tat-pyridoxine-5-P oxidase protein showed catalytic activity and was stable for 48 h. Moreover, the formation of pyridoxal-5-P was increased by adding exogenous Tat-pyridoxine-5-P oxidase to media pre-treated with the vitamin B6 precursor pyridoxine. In addition, the intracellular concentration of pyridoxal-5-P was markedly increased when Tat-pyridoxal kinase was transduced together with Tat-pyridoxine-5-P oxidase into cells.These results suggest that the transduction of Tat-pyridoxine-5-P oxidase fusion protein presents a means of regulating the level of pyridoxal-5-P and of replenishing this enzyme in various neurological disorders related to vitamin B6.

Animals↗
PubMed1998

Inhibition of xanthine oxidase by Puerto Rican plant extracts.

OBJECTIVES: This study was conducted to search for xanthine oxidase inhibitors in natural products obtained from plants collected in Puerto Rico and to assess the influence of these extracts in the prevention of cataractogenesis. BACKGROUND: Allopurinol is currently a xanthine oxidase inhibitor used in the treatment of gout. New alternatives with increased therapeutic activity and less side effects should be investigated. Preclusion of cataractogenesis in diabetic rats is also the focus of this investigation. Natural products in the form of plant extracts from Puerto Rico offer a rich and relatively untapped source for the discovery of new drugs that may address these kind of problems. METHODS: Nineteen collections of Myrtaceae plant extracts were screened for xanthine oxidase inhibition. A spectrophotometrical method was used employing allopurinol as positive control and a blank as negative control. A protocol of the assay with slight modifications was followed from the literature. Two extracts with the highest percentages of xanthine oxidase inhibition were evaluated for possible prevention of cataractogenesis in streptozotocin diabetic rats. The animals were given to drink these plant extracts ad libitum for three months while controls received water. The appearance of cataracts was assessed physically. RESULTS: Two of the nineteen plant extracts showed high inhibition percentages of xanthine oxidase. Eucalyptus deglupta and Syzygium malaccense displayed 51% and 64% inhibitions (IC50 44.5 micrograms/ml and IC50 51 micrograms/ml), respectively. As for the cataractogenesis inhibition, laboratory animals that drank E. deglupta for three months did not develop cataracts. CONCLUSIONS: Two plant extracts provided positive results with varying degrees of inhibition of xanthine oxidase. S. malaccense demonstrated the greatest xanthine oxidase inhibitory activity whereas E. deglupta presented the best finding for cataractogenesis prevention. The procedures used in this investigation are useful for the in vitro screening of xanthine oxidase inhibition and the in vivo evaluation of cataractogenesis prevention.

Allopurinol↗
PubMed1999

Regulation of lysyl oxidase, collagen, and connective tissue growth factor by TGF-beta1 and detection in human gingiva.

Gingival overgrowth is characterized by excess extracellular matrix accumulation and elevated levels of cytokines, including transforming growth factor-beta1 (TGF-beta1). The functional relationships between altered cytokine levels and extracellular matrix accumulation have not been extensively investigated in gingival cells and tissues. Lysyl oxidase catalyzes the final known enzymatic step required for cross-linking collagen and elastin in the synthesis of a functional extracellular matrix. This study investigated the regulation by TGF-beta1 of lysyl oxidase and its collagen and elastin substrates in early passage human gingival fibroblasts. In addition, TGF-beta1 regulation of connective tissue growth factor (CTGF) was assessed in human gingival cells and tissues. The results show that TGF-beta1 increases lysyl oxidase enzyme activity and mRNA levels for lysyl oxidase and alpha-1-type I collagen, but not elastin, in a dose- and time-dependent manner. Maximal stimulation of lysyl oxidase activity and mRNA levels for both lysyl oxidase and collagen occurs after 48 hours of treatment of gingival fibroblastic cells with 400 pM of TGF-beta1. This study shows for the first time that CTGF mRNA and protein are strongly and rapidly induced by TGF-beta1 in human gingival fibroblasts. Exogenous addition of 1 to 50 ng/ml CTGF to gingival fibroblasts stimulates production of lysyl oxidase enzyme activity up to 1.5-fold after 48 hours, and 50 ng/ml CTGF stimulated insoluble collagen accumulation 1.5- to 2.0-fold after 4, 11, and 18 days of treatment. It is interesting to note that the addition of CTGF-blocking antibodies in the presence of TGF-beta did not block TGF-beta stimulation of collagen mRNA levels. Thus, although CTGF itself contributes to increased insoluble collagenous extracellular matrix accumulation, CTGF does not mediate all of the effects of TGF-beta1 on stimulation of collagen mRNA levels in human gingival fibroblasts. Immunohistochemistry studies of gingival overgrowth tissue samples indicate for the first time detectable levels of CTGF protein in Dilantin-induced hyperplasia tissues also positive for TGF-beta1. CTGF was not found in TGF-beta1-negative samples. In addition, extracellular lysyl oxidase protein was detected in vivo. Taken together, these studies support mostly independent roles for TGF-beta1 and CTGF in stimulating collagenous extracellular matrix accumulation in human gingival fibroblasts and tissues.

Antibodies↗
PubMed1992

The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria.

Human monoamine oxidase A that had been synthesized in a reticulocyte lysate translation system was capable of binding to and inserting into either rat liver mitochondria or isolated mitochondrial outer membranes. The inserted form was as resistant to proteinase K as endogenous mitochondrial monoamine oxidase A. The insertion, but not the binding, of monoamine oxidase A was prevented by depleting the reaction mixture of either ATP (with apyrase) or ubiquitin (with purified antibodies against this polypeptide). Addition of ATP or ubiquitin, respectively, to these depleted mixtures restored the insertion of the enzyme. In the absence of mitochondria, in vitro synthesized monoamine oxidase A did not catalyze its own alkylation by the mechanism-based inhibitor, [3H]clorgyline. However, both monoamine oxidase A that had been membrane-inserted in vitro and monoamine oxidase A that had been bound to the mitochondria under conditions of ATP depletion catalyzed adduct formation. Furthermore, reaction of either clorgyline or another mechanism-based inhibitor, pargyline, with the membrane-bound enzyme during ATP depletion inhibited the insertion of monoamine oxidase A when ATP was restored. These observations indicate that monoamine oxidase A acquired a catalytically active conformation on interaction with the mitochondrial outer membranes prior to its ATP and ubiquitin-dependent insertion into the membrane.

Adenosine Triphosphate↗
PubMed1992

The complete derived amino acid sequence of human lysyl oxidase and assignment of the gene to chromosome 5 (extensive sequence homology with the murine ras recision gene).

Lysyl oxidase catalyzes the oxidation of lysine residues to alpha-aminoadipic-delta-semialdehyde. This is the first step in the covalent cross-linking of collagen and tropoelastin and results in the formation of insoluble collagen and elastic fibers in the extracellular matrix. We have characterized the complete nucleotide sequence of human lysyl oxidase (EC 1.4.3.13) and compared the derived amino acid sequence (417-amino acids) to rat lysyl oxidase and the mouse ras recision gene (rrg). 88% of amino acids and 83% of nucleotides were conserved between human and rat lysyl oxidase. The mouse ras recision gene demonstrated 89% conservation of amino acids with human lysyl oxidase. The sequence conservation was not evenly distributed along the molecule. The carboxy terminus of the protein, which contains the putative copper binding sites and is likely to be the catalytically active domain, was more highly conserved than the amino terminus. The 89% amino acid sequence similarity between the murine ras recision gene and human lysyl oxidase suggests that they are the same gene product. Therefore, in addition to cross linking of extracellular matrix proteins, lysyl oxidase may have a direct role in tumor suppression. Northern blot analysis of poly A+RNA from cultured skin fibroblasts revealed at least three-distinct transcripts, sized 4.8 kb, 3.8 kb and 2.0 kb. In addition, using a panel of human mouse cell hybrids, the lysyl oxidase gene was assigned to human chromosome 5.

Amino Acid Sequence↗
PubMed1991

Serum lysyl oxidase activity in chronic liver disease in comparison with serum levels of prolyl hydroxylase and laminin.

Lysyl oxidase was partially purified from serum by a diethylaminoethyl batch procedure in the presence of 6 mol/L urea and dialyzed against 3 mol/L KSCN. Using this method, we determined serum lysyl oxidase activity in 52 patients with liver disease and in 14 healthy controls, and we examined usefulness of serum lysyl oxidase in assessing liver fibrogenesis. For this purpose, serum lysyl oxidase activity in chronic liver disease was compared with serum levels of prolyl hydroxylase and laminin P1. As compared with controls, serum lysyl oxidase activity increased 1.6-fold in chronic persistent hepatitis, 4.4-fold in chronic active hepatitis and 11.8-fold in cirrhosis, indicating an increase in concert with the development of liver fibrosis. In hepatocellular carcinoma, the serum activity, although significantly increased, was lower than that in cirrhosis. Serum prolyl hydroxylase was significantly increased in chronic active hepatitis, in liver cirrhosis and in hepatocellular carcinoma. Serum laminin P1 was significantly increased in chronic active hepatitis, in cirrhosis and in hepatocellular carcinoma. Serum lysyl oxidase activity did not correlate significantly with serum levels of prolyl hydroxylase and laminin P1 in any subject or in any subgroup. The magnitude of the increase and the abnormal percentage of serum lysyl oxidase activity were larger than those for serum prolyl hydroxylase and laminin P1. These results suggest that serum lysyl oxidase activity is a more sensitive indicator of liver fibrosis than serum prolyl hydroxylase and laminin P1.

Adult↗
PubMed1977

Significance of multiple forms of brain monoamine oxidase in situ as probed by electron spin resonance.

Spin-labeled hydroxyamphetamine, a competitive reversible inhibitor of brain monoamine oxidase, has been shown to be useful as an electron spin resonance (ESR) probe of the microenvironment of the active sites of the possible monoamine oxidase multiple forms. The ESR spectrum of spin-labeled hydroxyamphetamine was strongly quenched upon binding to the enzyme. The conformation of the active site of rat brain monoamine oxidase existing in various physical states, i.e. monoamine oxidase in situ (intact brain mitochondria), crude solubilized monoamine oxidase (MAOS) and isolated monoamine oxidase fractions (MAOa and MAOb) were critically and systematically examined. Nonlinear least squares regression analyses have been used to fit the binding data (obtained at room temperature with varying spin-labeled hydroxyamphetamine concentrations) to three groups of independent noninteracting ligand-binding models. A Gibbs-Helmholtz relationship was applied to the interpretation of the measured apparent association constant K as a function of temperature ranging from 4-50 degrees with increments of 2 degreesmfrom the extracted intensive parameters, k (intrinsic association constant) and deltaF (intrinsic free energy), as well as the apparent heat, deltaH, it was clear that the microenvironment of the binding sites existing in the more purified enzyme fractions MAOa and MAOb were similar to those found in the crude solubilized enzyme. More importantly, they correlated well with the conformation of the sites characterized in situ. The data suggested that the microenvironment of this multienzyme system was unperturbed in spite of the treatment due to the isolation process. In terms of the composition of binding sites, MAOa appeared to be heterogeneous while MAOb appeared to be more homogeneous. Since the isolated fractions MAOa and MAOb possessed marked different substrate specificities, these observations directly implied that monoamine oxidase multiple forms do exist in situ. The extracted extensive parameters, n (specific binding activity, nanomoles/mg of protein), as well as the measured characteristic transition temperatures, indicated that the relative abundance of the sites which directly affected substrate specificities was indeed altered. The consistency of the characteristic transition temperatures of 21 degrees and 38 degrees for the case of intact membrane preparations was particularly significant. A tenable hypothesis is that the manipulation in the composition of the monoamine oxidase binding forms through intimate lipid-protein interactions, which has been amply demonstrated in many biomembrane systems to be functionally important might be the underlying regulatory mechanism in vivo.

Animals↗
PubMed1991

Stereochemical trends in copper amine oxidase reactions.

Copper amine oxidases (EC 1.4.3.6) exhibit atypical stereochemical patterns in the reactions they catalyze. Dopamine and tyramine are oxidized with abstraction of the pro-R hydrogen by the porcine plasma amine oxidase, the pro-S hydrogen by pea seedling amine oxidase and a net nonstereospecific proton abstraction by the bovine plasma enzyme. This provides the first example in which a reaction catalyzed by enzymes in the same formal class occurs by all three possible stereochemical routes. To assess the underlying mechanistic significance of this heterogeneity, we have established the stereochemical course of the oxidation of tyramine by five additional copper amine oxidases using 1H NMR spectroscopy. Reactions catalyzed by rabbit and sheep serum amine oxidases are nonstereospecific. These enzymes exhibit rare mirror image binding with differential flux through two opposite and stereospecific reaction pathways. Differential primary kinetic isotope effects are observed for each mode, 8 and 4.6 for pro-S abstraction and 2.6 and 2.7 for pro-R abstraction by the sheep and rabbit amine oxidases, respectively. Tyramine oxidations catalyzed by the soybean and chick pea amine oxidases and porcine kidney diamine oxidase, however, are all stereospecific, occurring with loss of the pro-S hydrogen at C-1. Solvent exchange profiles are consistent within each stereochemical class of enzyme; the pro-R and nonstereospecific enzymes exchange solvent into C-2 of product aldehydes, the pro-S enzymes do not.

Amine Oxidase (Copper-Containing)↗
PubMed1989

Vicinal diamines as pyrroloquinoline quinone-directed irreversible inhibitors of lysyl oxidase.

The observation that aliphatic diamines become poor substrates as the carbon chain length decreases and that ethylenediamine, the shortest diamine, is an irreversible inhibitor of lysyl oxidase led to the investigation of the mechanism of inhibition by ethylenediamine. The cis but not the trans isomer of 1,2-diaminocyclohexane was also a potent irreversible inhibitor of lysyl oxidase, consistent with the interaction of both amino groups of vicinal diamines with an enzyme moiety. Both cis-1,2-diaminocyclohexane and ethylenediamine but not trans-1,2-diaminocyclohexane markedly perturbed the spectrum of free pyrroloquinoline quinone (PQQ), a covalently linked form of which is the carbonyl cofactor of lysyl oxidase. cis-1,2-Diaminocyclohexane also induced similar changes in the spectrum of lysyl oxidase. The perturbations of the spectra of PQQ or of lysyl oxidase by cis-1,2-diaminocyclohexane or ethylenediamine as well as the development of irreversible inhibition of the enzyme by cis-1,2-diaminocyclohexane or ethylenediamine were all markedly reduced under anaerobic conditions. Moreover, approximately 1 mol of H2O2 was released per mol of PQQ or lysyl oxidase upon aerobic incubation with cis-1,2-diaminocyclohexane, while approximately 2 mol of 3H+ were released from cis-[1,2-3H] 1,2-diaminocyclohexane per mol of PQQ or lysyl oxidase under corresponding conditions. A proposal for the mechanism of inhibition of lysyl oxidase by vicinal diamines is presented which involves limited oxidation of the diamine linked to PQQ at the active site so that the PQQ-diamine complex is finally stabilized by a conjugated 6-membered ring.

Amino Acid Oxidoreductases↗
PubMed1987

Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein.

Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.

Cell-Free System↗
PubMed1987

Comparison of subcellular activation of the human neutrophil NADPH-oxidase by arachidonic acid, sodium dodecyl sulfate (SDS), and phorbol myristate acetate (PMA).

The phorbol myristate acetate (PMA) stimulation of the human neutrophil NADPH-oxidase has been demonstrated through the activation of protein kinase C (PK-C), using light membrane fractions from nitrogen-cavitated cells. Both arachidonic acid (AA) and sodium dodecyl sulfate (SDS) can also generate an active oxidase in cellfree systems. That the source of O2- with AA and SDS activation is the same NADPH-oxidase as previously studied was confirmed by the similar pH optima and Km values for NADPH as those previously described for the O2- -generating activity harvested from pre-stimulated human neutrophils. In contrast to the stimulation by PMA, however, the stimulation of the NADPH-oxidase by AA and SDS does not appear to require protein kinase C activation: the action of AA and SDS is independent of the addition of PK-C cofactors to the system, and the inhibitor of PK-C activity, H-7, had no effect on the stimulation by AA or SDS. AA and SDS activation are comparable, but the level of NADPH-oxidase expression is sixfold greater with each of these agents than that obtained with a reconstituted PK-C system. The basis of this difference in oxidase expression is unclear, but these findings suggest strongly that although activated PK-C is capable of stimulating a dormant NADPH-oxidase in a cellfree system, this is not the sole pathway for oxidase activation.

Arachidonic Acid↗
PubMed1986

Significance of xanthine oxidase in capillary endothelial cells.

Antibodies to xanthine oxidase from bovine milk lipid globules localize the antigen in capillary endothelial cells of many tissues including liver, heart, lung and kidney, but not in other epithelial, endothelial or mesenchymal cell types. The antigen from bovine capillaries was purified by immunoaffinity chromatography and shown by chemical, enzymatic and immunological methods to be indistinguishable from milk xanthine oxidase. Using an ultrasensitive radioimmunoassay, concentrations of this protein were found to be 1 000-10 000-fold higher in capillary endothelial cells than in other cells studied except mammary epithelial cells which were also rich in xanthine oxidase. Similar results were obtained with human cells and tissues. In the cytoplasm of capillary endothelial cells, xanthine oxidase was present as a dehydrogenase which was rapidly converted to the O-2-radical-producing oxidase form after release by cell disrupture. This conversion was partly prevented by addition of thiol reagents. Free xanthine oxidase was not detected in human serum, even from patients with extensive capillary lesions. However, specific-apparently constitutive--antibodies (IgG) were present at high concentrations (1-8% of total IgG) in the sera of all individuals tested. A role of these specific antibodies in the removal of the potentially hazardous oxidase form of xanthine oxidase is discussed.

Adenocarcinoma↗
PubMed1984

The NADPH oxidase of human polymorphonuclear leukocytes. Evidence for regulation by multiple signals.

Activation of the membrane-bound NADPH oxidase in human polymorphonuclear leukocytes can be triggered by chemoattractants, the tumor promoter phorbol myristate acetate or the calcium ionophore A23187. We have shown previously that these stimuli have markedly different temporal patterns of oxidase activation (McPhail, L. C., and Snyderman, R. (1983) J. Clin. Invest. 72, 192-200), suggesting that each follows, at least in part, a unique transductional pathway. We now report that if leukocytes were sequentially exposed to any of several combinations of heterologous stimuli, the pattern of activation by the second stimulus was strikingly altered, resulting in a more rapid rate and enhanced level of oxidase activation by the second stimulus. This suggests that exposure of cells to the first stimulus (priming) had influenced an intermediate also used by the second stimulus. The signal for priming could be clearly distinguished from the signal causing oxidase activation by the dose-response curves for each, as well as by the use of several pharmacologic agents. In addition, if leukocytes were given sequential doses of homologous stimuli, either partial (phorbol myristate acetate) or full (N-formyl-methionyl-leucyl -phenylalanine and A23187) desensitization of oxidase activation was observed. These results demonstrate that these stimuli share a common intermediate in the pathway of oxidase activation. Moreover, the data indicate that NADPH oxidase activation is regulated by at least three distinct signals: signal 1 (priming), signal 2 (activation), and signal 3 (inactivation). It is likely that more than one intracellular messenger exerts a modulating influence on NADPH oxidase activity and that its regulation involves the interplay between several cellular control proteins.

Calcimycin↗
PubMed1983

Selective effects on catalysis by the multiple forms of monoamine oxidase produced by interactions of acidic phospholipids with mitochondrial membranes.

The effect of acidic phospholipids on the A and B multiple forms of membrane-bound mitochondrial monoamine oxidase has been investigated by incubating liposomes with isolated rat liver mitochondrial outer membrane preparations at lipid:protein ratios of 0.01 to 1. A strong inhibition of monoamine oxidase B was observed with phosphatidylserine and a moderate activation of monoamine oxidase A with phosphatidylinositol, while cardiolipin had no significant effect on either form. The specificity of phosphatidylserine inhibition for monoamine oxidase B was also confirmed in mitochondrial outer membrane isolated from tissues containing exclusively the A or B form of the enzyme (human placenta and bovine liver). Levels of incorporation were comparable for all the phospholipids and tissues studied and could not account for the different effects observed. Inhibition of monoamine oxidase B was found to be similar in an intact mitochondria preparation to that observed in the isolated outer membrane. A recent report of activation of both monoamine oxidase forms in delipidated whole mitochondria by the acidic phospholipids was re-examined and found to involve release of monoamine oxidase from the mitochondria. The details of the effects of phosphatidylserine and phosphatidylinositol on membrane-bound monoamine oxidase are consistent with the concept of the multiple forms as two distinct peptides, and suggest a second possible mode of in vivo regulation of substrate specificity.

Animals↗
PubMed1994

Reaction of 5-ethynyluracil with rat liver xanthine oxidase.

5-Ethynyluracil is a time-dependent and tight binding inhibitor of xanthine oxidase. The maximal value of the first-order rate constant for onset of inhibition is 0.01 s-1, and the concentration of 5-ethynyluracil which gives one-half of this value is 190 microM. Because the t1/2 for formation of active enzyme from inhibited enzyme is greater than 30 h in the absence of NADH, inhibition of xanthine oxidase by 5-ethynyluracil is functionally irreversible. One equivalent of 5-[2-14C]ethynyluracil/equivalent of active enzyme is required for complete inhibition. Allopurinol (100 microM), a potent inhibitor of xanthine oxidase, and cyanide (5 mM), an inactivator of the enzyme, do not abolish the binding of 5-[2-14C]ethynyluracil to the enzyme. Because radiolabel is released from 5-[2-14C]ethynyluracil-treated enzyme by treatment with 6 M guanidine HCl, a stable covalent bond is not formed between the inhibitor and the enzyme. However, the radiolabel released from inhibited enzyme is not 5-ethynyluracil. Moreover, NADH restores catalytic activity to the inhibited enzyme and displaces the radiolabel as 5-acetyluracil. Thermal denaturation of 5-ethynyluracil-inhibited xanthine oxidase results in the release of approximately equal amounts of 5-acetyluracil and a more hydrophilic product. Consequently, the 5-ethynyluracil-xanthine oxidase complex yields different degradation products of 5-ethynyluracil under different denaturation conditions. Seven uracil analogues with 5-substituents were tested as time-dependent inhibitors of xanthine oxidase. 5-Ethynyluracil is the only uracil analogue that potently inhibited xanthine oxidase. The reactivity of these uracil derivatives with sulfite was also determined. 5-Ethynyluracil is many fold more susceptible to nonenzymatic nucleophilic addition of sulfite than are the other analogues. Thus, the potency of these uracil analogues as inhibitors of xanthine oxidase is related to the nonenzymatic reactivity of the analogues with sulfite.

Allopurinol↗
PubMed1994

Effects of sonicated eosinophils on the in vitro sensitivity of human lymphoma cells to glucose oxidase.

We report here that cultured human lymphoma cells in the absence of sonicated eosinophils are sensitive to killing by glucose oxidase (beta-D-glucose:oxygen-oxido reductase; EC 1.1.3.4) at concentrations as low as 0.025 microgram/ml, a level that can be rapidly attained in s.c. tumor implants in mice that receive a single nonlethal injection of enzyme. Multiple clonogenic assays were used to measure the survival of human lymphoma cell lines (H9 and ARH-77) cultured for 14 days in complete RPMI 1640 supplemented with exogenous glucose oxidase (0.025-2.5 micrograms/ml) or an immunoconjugate containing glucose oxidase (0.25-25 micrograms/ml) in the presence or absence of catalase (10 micrograms/ml) or an equal number of sonicated human eosinophils with or without supplemental 100 microM Br-, I-, or SCN-. In addition, we used an immunoassay to measure the concentration of glucose oxidase in s.c. implants of the Sp 2/0 myeloma tumor at 0-30 min after an i.v. injection of 50 micrograms of enzyme into 21 BALB/c mice. Doses of glucose oxidase as small as 0.025 microgram/ml killed more than 3 logs of tumor cells. Catalase completely inhibited, and sonicated human eosinophils partially inhibited, the killing by glucose oxidase or immunoconjugate, whereas supplemental halides had no effect. Glucose oxidase i.v. produced levels > 0.04 microgram/g of tumor for 30 min after injection with a peak concentration of 0.079 microgram/g of tumor within 5 min of injection. These results are important because certain human lymphomas contain extensive extracellular deposits of eosinophil peroxidase, thereby making these tumors potentially less susceptible to killing by otherwise therapeutic doses of glucose oxidase.

Drug Screening Assays, Antitumor↗