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PubMed1990

Structural and catalytic properties of copper in lysyl oxidase.

The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.

2,2'-Dipyridyl↗
PubMed1990

Evaluation of the role of xanthine oxidase in myocardial reperfusion injury.

The free radical-generating enzyme xanthine oxidase has been hypothesized to be a central mechanism of the injury which occurs in postischemic tissues; however, its importance remains controversial. Much attention has focused on the role of this enzyme in myocardial reperfusion injury. While xanthine oxidase has been observed in ischemic tissue homogenates, the presence and importance of radical generation by the enzyme in intact tissues are unknown. Therefore, we performed electron paramagnetic resonance, nuclear magnetic resonance and hemodynamic studies to measure the presence and significance of xanthine oxidase-mediated free radical generation in the isolated rat heart. When isolated perfused rat hearts were reperfused after 30 min of global ischemia, myocardial function and coronary flow were significantly improved in the presence of the definitive xanthine oxidase blocker oxypurinol. Free radical concentrations measured by spin-trapping with 5,5'-dimethyl-1-pyrroline-N-oxide were significantly decreased by oxypurinol and the energetic state of the heart was improved as reflected by an increased recovery of phosphocreatine and a higher phosphocreatine/Pi ratio. ATP recovery, however, was not altered, indicating that the improved functional and metabolic state of the heart was not due to ATP salvage. Spectrophotometric assays for the enzyme showed an increase in the amount of xanthine oxidase relative to dehydrogenase following ischemia, and a total available xanthine oxidase pool in the rat heart of approximately 150 milliunits/g of protein. Thus, xanthine oxidase is a significant source of the oxidative injury which occurs upon reperfusion of the ischemic rat heart.

Animals↗
PubMed1990

Immunochemical and electrophoretic analyses of phosphorylated native and recombinant neutrophil oxidase component p47-phox.

Human polymorphonuclear neutrophils (PMNs) possess a potent oxygen-dependent microbicidal system that depends on the activity of a stimulus-activated multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Patients with chronic granulomatous disease (CGD) lack activity of this oxidase and consequently suffer severe and frequent infections. Components of the oxidase include both membrane-bound factors (most notably, cytochrome b559, which is absent in the X-linked form of CGD) and at least two cytosolic factors, one or the other of which is absent in autosomal CGD. Patients with CGD, particularly the autosomal type, have defective phosphorylation of proteins in the 44 to 48 Kd range. A polyclonal antiserum (B-1) that recognizes cytosolic oxidase components of 47 and 67 Kd was used to identify phosphoproteins in a cell-free oxidase system. Two-dimensional gel electrophoresis showed the identity of the 47-Kd cytosolic protein (p47-phox) recognized by B-1 and the cationic 47-Kd protein that is phosphorylated in normal but not p47-phox-deficient CGD cytosol during activation of the NADPH-dependent oxidase. All full-length and C-terminal recombinant p47-phox proteins augmented the superoxide-generating capacity of the cell-free system and were phosphorylated when added to cytosol from normal subjects or from a patient with p47-deficient autosomal CGD. These studies provide compelling evidence that the 47-Kd cationic protein that is a substrate for phosphorylation during the activation of PMNs is, in fact, p47-phox, a cytosolic protein previously shown to be critical for normal activity of the NADPH-dependent oxidase of PMNs.

Autoradiography↗
PubMed1990

Bis(benzyl)polyamine analogs as novel substrates for polyamine oxidase.

N,N'-Bis(benzyl)polyamine analogs were found to be substrates for highly purified polyamine oxidase. Metabolism of these analogs was apparently dependent on molecular O2 and resulted in the formation of benzaldehyde, H2O2, and a polyamine analog with free terminal amines. The debenzylation reaction was optimal between pH 9 and 10, identical to the pH optimum for polyamine oxidase activity when N1-acetylspermine was used as the substrate. On a molecular sieve column the debenzylating activity co-eluted with N1-acetylspermine oxidizing activity, at an apparent molecular mass of approximately 65 kDa. The purified enzyme also appeared to have a molecular mass of approximately 65 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Debenzylation of the bis(benzyl)polyamines was competitively inhibited by N1-acetylspermine and N1-acetylspermidine. The specific irreversible inhibitor of polyamine oxidase, N1,N4-bis(buta-2,3-dienyl)butanediamine also inhibited the debenzylation, whereas inhibitors of diamine and monoamine oxidases did not. The evolution of benzaldehyde from bis(benzyl)polyamine analogs by polyamine oxidase allowed the development of a simple rapid spectrophotometric assay for use in the measurement of polyamine oxidase activity in partially purified tissue or cell extracts. Further, metabolism of a bis(benzyl)polyamine analog by polyamine oxidase was found to be an important element in the growth inhibitory properties of the compound in a mouse model of malaria.

Animals↗
PubMedDate not supplied

[Amine oxidases of the human placenta].

Some current advances in studies of human placenta amine oxidases monoamine oxidase (MAO), diamine oxidase (DAO) and benzyl amine oxidase are reviewed. Localization of these enzymes in placental tissue as well as distribution in subcellular fractions (mitochondria, microsomes and cytosol) are considered. Presence of multiple forms of MAO in placenta is discussed: three types of MAO A, B and B' were found in human placenta, while MAO of the B' type was not detected in other mammalian tissues. Main physicochemical and catalytic properties of human placenta amine oxidases are considered as well as some cases of alterations in properties of the amine oxidases during anomalous pregnancy are exhibited. Physiological role of MAO and DAO in human placenta as well as use of the amine oxidase test in estimation of the state of pregnancy are discussed.

Amine Oxidase (Copper-Containing)↗
PubMed1989

Lipid binding by Escherichia coli pyruvate oxidase is disrupted by small alterations of the carboxyl-terminal region.

Escherichia coli pyruvate oxidase is a membrane-associated flavoprotein dehydrogenase which is greatly activated by lipids and detergents. The carboxyl-terminal region of the protein has been shown to play a critical role in the interaction with lipids. We report mutations generated by chemical and oligonucleotide-mediated site-directed mutagenesis of the poxB gene which result in enzymes defective in lipid activation. Nine mutants were isolated which encode enzymes with point mutations in the carboxyl-terminal segment of the protein. Two mutant lesions introduced termination codons giving enzymes lacking the last nine or three amino acids of the protein which were unable to interact with detergents in vitro and were unable to function in vivo. Of the missense mutants isolated, two were most informative. One was the substitution of Glu-564 with proline in the PoxB16 oxidase. This residue lies in the center of a putative lipid-binding amphipathic alpha-helix (Arg-558 to Thr-568) located close to the carboxyl terminus. Strains producing the PoxB16 oxidase were devoid of oxidase activity in vivo, the enzymes could not be activated by Triton X-100, and were activated poorly by phospholipids in vitro. These results indicated that the PoxB16 oxidase lacked normal lipid-binding ability. Another mutant oxidase (PoxB15) in which proline was substituted for Asp-560 at the beginning of the amphipathic alpha-helix had normal oxidase activity. These findings indicate that the amphipathic alpha-helix structure plays an essential role in the activation and lipid-binding properties of the enzyme. The second informative missense mutation was the substitution of the carboxyl-terminal arginine with glycine. This enzyme showed normal activation in vitro by phospholipids and some detergents, and somewhat reduced activity in vivo. This mutant enzyme appeared to dissociate from detergent vesicles more readily than does the normal enzyme. A model for the membrane interaction of the carboxyl terminus based on the properties of these mutant proteins is presented.

Alleles↗
PubMed1985

Thermodynamic control of D-amino acid oxidase by benzoate binding.

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.

Animals↗
PubMed1986

Reaction pathway of bovine aortic lysyl oxidase.

The catalysis of amine oxidation by lysyl oxidase has been probed to assess for the likely order of substrate binding and product release and to discriminate between mechanistic alternatives previously proposed for other copper-dependent amine oxidases using molecular oxygen as a substrate. Lineweaver-Burk plots revealed a pattern of parallel lines when the oxidation of n-butylamine was followed at different fixed concentrations of oxygen consistent with a "ping-pong" kinetic mechanism in which the aldehyde is produced and released before the binding of oxygen, the second substrate. Initial burst experiments revealed the ability of lysyl oxidase to form and release n-butyraldehyde in amounts stoichiometric with functional active site content in the absence of oxygen, consistent with the ping-pong kinetics obtained. Reciprocal plots of n-butylamine oxidation in the presence of fixed concentrations of the reaction products were consistent with a Uni Uni Uni Bi ping-pong kinetic mechanism with the aldehyde being the first, H2O2 the second, and ammonia the last departing product. Moreover, spectral studies of the oxidation of p-hydroxybenzylamine by lysyl oxidase indicated that the enzyme does not process the amine substrate to a noncovalently bound p-hydroxybenzaldimine intermediate subsequently to be hydrolyzed to p-hydroxybenzaldehyde. The kinetic mechanism of lysyl oxidase thus appears to be similar to those described for diamine oxidase and pig plasma monoamine oxidase.

Aldehydes↗
PubMed1985

Activation of mouse macrophages causes no change in expression and function of phorbol diesters' receptors, but is accompanied by alterations in the activity and kinetic parameters of NADPH oxidase.

Mouse peritoneal macrophages activated in vivo by the injection of Corynebacterium parvum release larger amounts of superoxide anion (O2-) than macrophages from control mice when stimulated with phorbol myristate acetate (PMA). The biochemical bases for this enhanced response of activated macrophages have been investigated by studying the expression and function of receptors for the stimulant, and the activity of the enzyme NADPH oxidase which is responsible for the production of O2- in leucocytes. Studies of binding of phorbol dibutyrate, an agent closely related to PMA, showed that the affinity constants (Kds) and the number of binding sites were the same in resident and activated peritoneal macrophages. The activity of the NADPH oxidase was, however, different in the two macrophage populations which differ in their capacity to release O2-. NADPH oxidase activity was studied in macrophage monolayers after lysis with deoxycholate. The main features of this activity were as follows: stimulation of macrophages with PMA or zymosan caused an increase in NADPH-dependent O2- production; NADPH oxidase activity in the lysates followed the same dose-response curve for different concentrations of PMA as O2- release by intact macrophages; O2- release by intact macrophages could be fully accounted for by NADPH-dependent O2- production by macrophage lysates; activity was strictly substrate-specific, in that NADH could not substitute for NADPH; after stimulation with PMA or zymosan, NADPH oxidase activity was higher in lysates of C. parvum-activated macrophages than in lysates of resident macrophages; NADPH oxidase activities of activated and resident macrophages differed markedly in their kinetic parameters. The NADPH oxidase of macrophages activated by C. parvum or trehalose dimycolate of mycobacterial origin displayed a five to seven times lower Km compared to the enzyme in resident macrophages.

Animals↗
PubMed1985

Enhancement by Ca2+ or Mg2+ of catalytic activity of the superoxide-producing NADPH oxidase in membrane fractions of human neutrophils and monocytes.

To examine the role of divalent cations in the generation of superoxide anion (O2-) by the NADPH oxidase system of phagocytic cells, membrane-rich fractions were prepared from human neutrophils and monocytes. O2- generation by the fractions in sucrose was enhanced by addition of Ca2+ or Mg2+. EDTA inhibited most of the O2- generation; Ca2+ or Mg2+ reversed the inhibition. Zn2+, Mn2+, or Cu2+ completely inhibited O2- production. Neutrophil membrane fraction solubilized with Triton X-100, then passed through a chelating column, lost 80% of its oxidase activity; the loss could be reversed by addition of Ca2+ or Mg2+. Addition of 0.3 mM Ca2+ or Mg2+ protected against thermal instability of the enzyme. Kinetic analysis of the neutrophil oxidase activity as a function of NADPH and Ca2+ or Mg2+ concentrations showed that cation did not interact with NADPH in solution or affect the binding of NADPH to the oxidase; rather, cation bound directly to the oxidase, or to some associated regulatory component, to activate the enzyme. For the neutrophil oxidase, the Km for NADPH was 51 +/- 6 (S.D.) microM. Hyperbolic saturation was observed with Ca2+ and Mg2+, and the Kd values were 1.9 +/- 0.3 and 2.9 +/- 0.3 microM, respectively, suggesting that the oxidase, or some associated component, has a relatively high-affinity binding site for Ca2+ and Mg2+.

Calcium↗
PubMed1987

Activation of neutrophil NADPH oxidase in a cell-free system. Partial purification of components and characterization of the activation process.

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is converted from an inactive to an active form upon stimulation of the neutrophil. This activation process was examined using a recently developed cell-free system in which dormant oxidase is activated by arachidonic acid in the presence of a soluble factor from the neutrophil (Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743). NADPH oxidase from unstimulated human neutrophils was detected only in the membrane fraction. The soluble activation factor was localized entirely to the cytosolic fraction and exhibited two peaks of activity when partially purified under nondenaturing conditions: a major peak with a molecular mass of approximately 250 kDa and a variable minor peak with a mass of approximately 40 kDa. Both forms activated NADPH oxidase in a similar manner and did not exhibit synergy when combined. The cytosolic factor is not protein kinase C (or another kinase) as both peaks of factor activity could be resolved from the protein kinase C peak and neither required calcium or ATP to activate the oxidase. Activation of NADPH oxidase did require the simultaneous presence of the membrane fraction, the cytosolic factor, arachidonic acid, and magnesium. Following activation, however, only the membrane fraction was then required for O2- production. Cytosolic factor levels were normal in five patients with either X-linked or autosomal recessive cytochrome b-negative chronic granulomatous disease. In contrast, the membrane fractions from each failed to generate O2-, indicating that the defects in these two genetic forms of chronic granulomatous disease reside either in the oxidase itself or in a membrane component required for activation.

Cell-Free System↗
PubMed1983

Activation of mouse peritoneal macrophages by lipopolysaccharide alters the kinetic parameters of the superoxide-producing NADPH oxidase.

Macrophages activated by infection or elicited by injection of lipopolysaccharide (LPS), when stimulated with phorbol myristate acetate, release greater amounts of superoxide anion (O-2) than do normal resident macrophages. This enhanced production of O-2 and of other oxygen metabolites derived from O-2 is responsible, at least in part, for the enhanced microbicidal activity of these cells. To investigate the molecular basis for the enhanced oxygen metabolite response, the kinetic parameters of the enzyme responsible for NADPH-dependent production of O-2 (NADPH oxidase) were compared in LPS-elicited and resident mouse peritoneal macrophages. Resident and LPS-elicited macrophages were stimulated with phorbol myristate acetate, disrupted by sonication, assayed for their content of NADPH, and fractionated by centrifugation. The distribution of NADPH oxidase activity among the fractions was similar in both types of macrophage. A membrane-rich 27,000 X g pellet, which had the highest specific activity for the oxidase among the various fractions, was utilized to study the kinetic parameters of the oxidase from normal and LPS macrophages. The oxidase from LPS-elicited macrophages displayed a higher Vmax and a lower Km for NADPH than did the oxidase from normal cells. LPS-elicited cells also had a higher intracellular concentration of NADPH than did normal cells. The altered Km and Vmax, combined with the higher concentration of NADPH, resulted in a 2.2- to 3.5-fold increase in the calculated velocity of the oxidase from LPS-elicited macrophages compared with that from resident macrophages. These results suggest that the enhanced oxygen metabolite response of activated macrophages is due, in part, to modification of the enzyme responsible for the production of O-2.

Animals↗
PubMed1984

Terminal oxidases of Escherichia coli aerobic respiratory chain. II. Purification and properties of cytochrome b558-d complex from cells grown with limited oxygen and evidence of branched electron-carrying systems.

Cytochrome b558-d complex, a terminal oxidase in the respiratory chain of Escherichia coli K12 grown under the condition of limited oxygen, was purified to near homogeneity. The purified oxidase complex is composed of equimolar amounts of two polypeptides, with Mr = 26,000 and 51,000, determined with gel electrophoresis in the presence of sodium dodecyl sulfate. It contains 12.3 nmol of protoheme, 9.54 nmol of cytochrome d, and 26.6 nmol of iron/mg of protein. The enzyme is a "cytochrome bd-type oxidase," which shows absorption peaks at 558 and 624 nm in the difference spectrum at 77 K. This oxidase combines with CO, and the Soret region of the CO difference spectrum at room temperature has a peak at 420 nm and troughs at 430 and 442 nm. The oxidation-reduction potentials of cytochrome b558 and cytochrome d at pH 7.4 were estimated to be +10 mV and +240 mV, respectively. The cytochrome b558-d complex catalyzes the oxidation of ubiquinol-1, menadiol, and ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride. This oxidase activity was inhibited by the respiratory inhibitors piericidin A, KCN, and NaN3, but the cytochrome b558-d complex was less sensitive to the inhibitors than the cytochrome b562-o complex. The Km values for oxygen of purified cytochrome b558-d complex and cytochrome b562-o complex were determined to be 0.38 and 2.9 microM, respectively. Formation of a membrane potential by the reconstituted cytochrome b558-d complex in liposomes was observed with the fluorescent dye 3,3'-dipropylthiocarbocyanine iodide on addition of ubiquinol-1. This is the first indication that there is a coupling site in the terminal oxidase, which contains cytochrome d. Steady state kinetics of cytochromes in the membrane showed that these oxidase complexes branch at the site of ubiquinone-8 in the respiratory chain. From these and previous results, branched electron-carrying systems of the E. coli respiratory chain are proposed.

Aerobiosis↗
PubMed1984

Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin.

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.

Allopurinol↗
PubMed1976

Monoamine oxidase inhibition and the induction of ponto-geniculo-occipital wave activity by reserpine in the cat.

Reserpine induces ponto-geniculo-occipital wave activity similar to that seen in the cat during the rapid eye movement phase of sleep. This action of reserpine was blocked by the monoamine oxidase inhibitors, pheniprazine, harmaline and clorgyline, but not deprenyl or its demethylated analog. After a single dose (20 mg/kg) of either pheniprazine or harmaline, the time course of antagonism of the effect of reserpine was in good correspondence with inhibition of monoamine oxidase but not with restoration of the serotonin content of several regions of the brain. Harmaline had a reversible effect while that of pheniprazine persisted for weeks. Clorgyline in low doses (0.5-1.0 mg/kg), at which it is a specific inhibitor of type A monoamine oxidase, antagonized the action of reserpine for over 2 weeks. Deprenyl and its demethylated analog failed to suppress the reserpine-induced waves even at 10 times the dose of clorgyline when they should have completely inhibited type B monoamine oxidase. In contrast to antagonism of reserpine, suppression of rapid eye movement sleep by these monoamine oxidase inhibitors could be temporally dissociated from their inhibition of the enzyme. It is concluded that suppression of the induction of ponto-geniculo-occipital waves by reserpine after administration of the monoamine oxidase inhibitors is a specific effect of these drugs and is related to inhibition of type A monoamine oxidase. Suppression of rapid eye movement sleep is probably a nonspecific effect and not related to inhibition of the enzyme.

Animals↗
PubMed1982

Diamine oxidase in the diagnosis of ruptured fetal membranes.

Diamine oxidase activity is present in amniotic fluid but absent from vaginal secretions, providing a method for diagnosing rupture of membranes. Vaginal secretions were absorbed onto sterile paper strips, eluted, and assayed for diamine oxidase activity. Interfering substances included maternal serum, iodine antiseptics, and large amounts of meconium. Two of 109 prenatal clinic (negative control) specimens displayed measurable diamine oxidase activity; one of 144 postamniotomy (positive control) specimens had no diamine oxidase activity. Of 75 test specimens obtained by speculum examination and 488 test specimens obtained without the aid of a speculum, 18 and 62, respectively, displayed a diamine oxidase test result different from results of conventional tests (nitrazine paper, ferning, fetal cell stains). These discrepancies are discussed in view of the utility of the diamine oxidase test as an adjunct to conventional methods for diagnosing rupture of membranes. The diamine oxidase test appears to be objective, sensitive, quantitative, and capable of detecting amniotic fluid leaks more than 25 hours after rupture of membranes. It can be performed in 1 hour and requires a scintillation counter.

Amine Oxidase (Copper-Containing)↗
PubMed1995

[An endogenous inhibitor of monoamine oxidase A (tribulin A) from brain: purification and structure identification].

The endogenous monoamine oxidase inhibitor, tribulin, contains several components which selectively (or nonselectively) inhibit monoamine oxidases A and B. The pig brain tribulin component selectively inhibiting monoamine oxidase A was purified and identified as 4-hydroxyphenylethanol using gas chromatography-mass spectrometry. This compound was also found in the rabbit brain tribulin fraction which selectively inhibits monoamine oxidase A but has no influence on monoamine oxidase B. 4-Hydroxyphenylethanol inhibits monoamine oxidase A in an incompetitive manner with respect to the substrate, serotonin (Ki = 1.4 mM). Possible pathways of 4-hydroxyphenylethanol synthesis and its biological importance as the monoamine oxidase A inhibiting component of tribulin are discussed.

Animals↗
PubMed1995

Functional reconstitution of the phagocyte NADPH oxidase by transfection of its multiple components in a heterologous system.

The phagocyte NADPH oxidase system, as previously defined by cell-free reconstitution, is comprised of five essential components, three of which are produced during late phagocytic differentiation--namely, two cytosolic proteins, p47- and p67-phox--and the large subunit of cytochrome b558, gp91-phox. To confirm that these are the only phagocyte-specific components necessary for oxidase activity in whole cells, the recombinant NADPH oxidase was reconstituted in a heterologous cell line. An undifferentiated multipotent leukemic cell line, K562, which expresses endogenous Rac and the small subunit of the flavocytochrome b558 (p22-phox), was cotransfected with episomal expression vectors containing cDNAs for the three other oxidase components. After 4 days of selection, the complete oxidase system was functionally reconstituted in transfected cells stimulated with phorbol myristate acetate or calcium ionophore. These easily transfected cells provide an ideal model system in which several oxidase components can be genetically manipulated and readily expressed. This system can be used to test the effects of mutations associated with any of the genes affected in chronic granulomatous disease and will facilitate studies on structure-function relationships within several oxidase components. This system will also aid in delineation of upstream regulators functioning through various signaling pathways.

Blotting, Western↗