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PubMed1997

Urate oxidase in prevention and treatment of hyperuricemia associated with lymphoid malignancies.

Standard prophylaxis and treatment of malignancy-associated hyperuricemia in the USA has been allopurinol with vigorous hydration, urinary alkalinization and osmotic diuresis. Urate oxidase, the enzyme that converts uric acid to allantoin (a readily excreted metabolite that has 5- to 10-fold higher solubility than uric acid), is an alternative therapy; however, few published findings support this practice. Between February 1994 and December 1996, we administered non-recombinant urate oxidase (Uricozyme) to 126 children with newly diagnosed non-B cell acute lymphoblastic leukemia (ALL) during the first 5 days of chemotherapy with methotrexate, 6-mercaptopurine or both. Their blood levels of uric acid and other indicators of tumor lysis were measured at diagnosis and during treatment and then compared with findings in 129 similarly treated historical controls who had received allopurinol to control hyperuricemia. Clinical responses to urate oxidase were also determined in eight patients with newly diagnosed B cell ALL or advanced-stage non-Hodgkin lymphoma. Patients treated with urate oxidase had rapid and significantly greater decreases in their blood uric acid levels than did the historical controls (median maximal level during treatment, 2.3 vs 3.9 mg/dl, P < 0.001). They also had lower creatinine (0.6 vs 0.7 mg/dl, P = 0.01) and blood urea nitrogen (11 vs 24 mg/dl, P < 0.001) levels. Similar findings were made in the eight cases of B cell ALL or non-Hodgkin lymphoma. None of the patients required dialysis for acute renal failure. Six (4.5%) of the 134 children given urate oxidase had allergic reactions, manifested primarily by urticaria, bronchospasm and hypoxemia. Thus, non-recombinant urate oxidase is a more effective uricolytic agent than allopurinol but is associated with acute hypersensitivity reactions, even in patients without a history of allergy.

Allopurinol↗
PubMed1997

Expression of NADPH oxidase is induced by all-trans retinoic acid but not by phorbol myristate acetate and 1,25 dihydroxyvitamin D3 in the human promyelocytic cell line NB4.

Human promyelocytic cells, NB4, differentiate into neutrophils in response to all-trans retinoic acid (ATRA). It has recently been proposed that NB4 cells have bilineage potential because these cells are also able to differentiate into monocyte/macrophages when exposed to a combination of 1,25-dihydroxyvitamin D3 (VD3) and phorbol myristate acetate (PMA). Differentiation of myeloid cells into neutrophils or monocytes is associated with the acquisition of the O2- producing enzyme, NADPH oxidase, which plays a critical role in microbial killing. In this study, the expression of the components of the NADPH oxidase complex during the differentiation of NB4 cells into neutrophils or macrophages has been investigated. Whereas cells exposed to ATRA were able to produce O2- after 2 days of differentiation, they remain unable to generate O2- when exposed to PMA or PMA + VD3. With the exception of p21rac, none of the other oxidase components was expressed in non-differentiated cells. Addition of ATRA induced the progressive expression and accumulation of p22phox, p91phox, p47phox and p67phox. Compared to the other components, p67phox was expressed late and its expression appeared to correlate most closely with the generation of O2- in the differentiation process. In PMA or PMA + VD3-differentiated NB4 cells, expression of the NADPH oxidase components was incomplete. Therefore, ATRA induced the expression of a functional NADPH oxidase complex in neutrophil-like NB4 cells. In contrast, when NB4 cells are exposed to monocytic differentiating agents, they acquire only part of the phenotypic characteristics of monocytes and lack one of the major phagocytic functionalities, the respiratory burst oxidase.

Calcitriol↗
PubMed2000

Novel glucose non-interference biosensor for lactose detection based on galactose oxidase-peroxidase with and without co-immobilised beta-galactosidase.

Two types of amperometric biosensors for lactose detection based either on co-immobilisation of two enzymes (galactose oxidase with peroxidase) or co-immobilisation of three enzymes (beta-galactosidase, galactose oxidase and peroxidase) were constructed. A graphite rod with pre-adsorbed ferrocene was used as a working electrode. The use of galactose oxidase instead of the frequently used glucose oxidase resulted in the construction of a glucose-non-interfering lactose sensor. Co-immobilisation of peroxidase with galactose oxidase allowed the effect of borate on the extension of the linear range and the effect of the working potential on galactose oxidase activation to be studied. The presence of beta-galactosidase greatly enhances the sensor's sensitivity, but its linear range is narrower than that of the sensor without beta-galactosidase. Addition of DEAE-dextran and inositol to the enzyme layer improved the half-life more than 16-fold compared with the sensor without stabilisers. A response time between 60 and 75 s (90% of the steady-state value) and a detection limit for lactose determination from 44 to 339 microM (signal-to-noise ratio = 3) were observed depending on the conditions. The precision of measurements of standard lactose solution for the trienzymatic and bienzymatic sensors was 2.19 and 2.02%, respectively. The precision of analysis of dairy products varied from 0.24 to 5.24%. Analyses of real samples showed good correlation with HPLC analysis; eight samples and 10 standard lactose solutions without pre-treatment were analysed in 1 h.

Animals↗
PubMed2005

Expression of functionally phagocyte-type NAD(P)H oxidase in pericytes: effect of angiotensin II and high glucose.

BACKGROUND INFORMATION: A growing body of evidence demonstrates the involvement of the oxidative stress in the development of vascular complications associated with diabetes, such as hypertension, retinopathy, nephropathy, neuropathy and atherosclerosis. However, the molecular mechanisms accountable for the increased production of reactive oxygen species (ROS) remain uncertain. Among others, the NAD(P)H oxidase is one of the most important sources of superoxide anion (O2-) that induce dysfunction of vascular cells. Pericytes (PCs) have an essential role in the capillary dysfunction in retinopathy and other vascular complications in diabetes. We questioned whether PCs express a functional phagocyte-type NAD(P)H oxidase, and examined the role of angiotensin II and high glucose on the activity of the oxidase complex and expression of the essential subunit p22(phox). RESULTS: The mRNA expression of p22(phox), p47(phox), p67(phox) and NOX 1 subunits, and the lack of gp91(phox) component, were detected in PCs by reverse transcriptase PCR. Western-blotting analysis demonstrated the protein expression of p22(phox), p47(phox) and p67(phox) subunits. As compared with the normal condition, stimulation of PCs with angiotensin II or high glucose induced: (i) an increase in ROS production and NAD(P)H oxidase activity, and (ii) an up-regulation of p22(phox) mRNA and protein expression. CONCLUSIONS: Taken together, the present study provides the first evidence that PCs express a functional phagocyte-type NAD(P)H oxidase, which is up-regulated by both angiotensin II and high glucose. Given the importance of ROS in vascular physiology and pathology, the NAD(P)H oxidase complex could be an important therapeutic target in the treatment of microvascular disorders.

Angiotensin II↗
PubMed1967

The inactivation of pea-seedling diamine oxidase by peroxidase and 1,5-diaminopentane.

1. The rate of oxidative deamination of 1,5-diaminopentane by pea-seedling extracts, which contain diamine oxidase [diamine-oxygen oxidoreductase (deaminating), EC 1.4.3.6], was increased by adding pyridoxal or pyridoxal phosphate. 2. Evidence was obtained that pyridoxal does not activate the apoenzyme of diamine oxidase, but prevents the inactivation of the enzyme. 3. This inactivation only occurred when 1,5-diaminopentane was the substrate and depended on a second thermolabile factor in the extract besides the diamine oxidase. 4. Purified diamine oxidase, when catalysing the oxidation of 1,5-diaminopentane, was rapidly inactivated in the presence of peroxidase. 5. The inactivation was prevented not only by pyridoxal and pyridoxal phosphate but also by several unrelated compounds including alpha-oxoglutarate, catechol and o-aminobenzaldehyde. 6. It is suggested that peroxidase catalyses the further oxidation of the product of the oxidative deamination of 1,5-diaminopentane to a compound that inactivates diamine oxidase. 7. The results diminish the relevance of previous evidence that plant diamine oxidase contains pyridoxal phosphate.

Aldehydes↗
PubMed1972

Inhibition of monoamine oxidase by substituted hydrazines.

1. The initial rate of inhibition of monoamine oxidase by phenethylhydrazine was shown to be similar, in pH-dependence and kinetic properties, to the oxidation of that compound by monoamine oxidase. 2. The time-course of irreversible inhibition of monoamine oxidase by phenethylhydrazine lags behind that of reversible inhibition. 3. Hydralzine was shown to be a reversible competitive inhibitor of monoamine oxidase, but phenylhydrazine is an irreversible inhibitor. Inhibition by the latter compound is not affected by the absence of oxygen, and the presence of substrate exerts no protective action. 4. Hydrazine does not inhibit monoamine oxidase unless a substrate and oxygen are present. 5. Phenethylidenehydrazine was found to be a time-dependent inhibitor of monoamine oxidase and the rate of inhibition was hindered by increasing oxygen concentration. 6. A mechanism for the inhibition of the enzyme by phenethylhydrazine is proposed in which the product of oxidation of this compound is a potent reversible inhibitor and an irreversible inhibitor of the enzyme. A computer simulation of such a mechanism predicts time-courses of inhibition that are in reasonable agreement with those observed experimentally.

Animals↗
PubMed2002

Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin.

During polyamine catabolism, spermine and spermidine are first acetylated by spermidine/spermine N(1)-acetyltransferase (SSAT) and subsequently oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine, respectively. In attempting to clone the PAO involved in this back-conversion pathway, we encountered an oxidase that preferentially cleaves spermine in the absence of prior acetylation by SSAT. A BLAST search using maize PAO sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma: the encoded proteins differed by 20 amino acids. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by 75% while spermidine and N (1)-acetylspermidine pools increased, suggesting that spermine was selectively and directly oxidized by the enzyme. Substrate specificity using lysates of oxidase-transfected HEK-293 cells revealed that the newly identified oxidase strongly favoured spermine over N (1)-acetylspermine and that it failed to act on N (1)-acetylspermidine, spermidine or the preferred PAO substrate, N (1), N (12)-diacetylspermine. The PAO inhibitor, MDL-72,527, only partially blocked oxidation of spermine while a previously reported PAO substrate, N (1)-( n -octanesulphonyl)spermine, potently inhibited the reaction. Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated spermine oxidase in order to distinguish it from the PAO involved in polyamine back-conversion. The identification of an enzyme capable of directly oxidizing spermine to spermidine has important implications for understanding polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically relevant polyamine analogues and inhibitors.

Amino Acid Sequence↗
PubMed2006

Homocysteine stimulates phosphorylation of NADPH oxidase p47phox and p67phox subunits in monocytes via protein kinase Cbeta activation.

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.

Calcium Channel Blockers↗
PubMed2007

Cross-talk between IRAK-4 and the NADPH oxidase.

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Amino Acid Sequence↗
PubMed1984

Comparison of the degradative fate of monoamine oxidase in endogenous and transplanted mitochondrial outer membrane in rat hepatocytes. Implications for the cytomorphological basis of protein catabolism.

The degradative fate of monoamine oxidase in endogenous and transplanted mitochondrial outer membrane has been compared in rat hepatocyte monolayers. Monoamine oxidase was specifically irreversibly radiolabelled by the suicide inhibitor [3H]pargyline. Hepatocyte monolayers were cultured in conditions in which rates of protein catabolism like those in vivo are maintained [Evans & Mayer (1983) Biochem. J. 216, 151-161]. Incubation of hepatocyte monolayers for 17 h with [3H]pargyline specifically radiolabels mitochondrial monoamine oxidase, as shown by Percoll-gradient fractionation of broken hepatocytes. Monoamine oxidase is degraded at a similar rate to that observed in liver in vivo (t1/2 approx. 63 h). The effects of leupeptin, methylamine and colchicine on the degradation of endogenous radiolabelled enzyme has been studied over prolonged culture periods. Culture of hepatocytes for periods of up to 80 h with inhibitors was not cytotoxic, as demonstrated by measurements of several intrinsic biochemical parameters. Leupeptin, methylamine and colchicine inhibit the degradation of endogenous monoamine oxidase by 60, 38 and 18% respectively. Monoamine oxidase in mitochondrial-outer-membrane vesicles introduced into hepatocytes by poly(ethylene glycol)-mediated vesicle-cell transplantation is degraded at a similar rate (t1/2 55 h) to the endogenous mitochondrial enzyme. Whereas leupeptin inhibits the degradation of endogenous and transplanted enzyme to a similar extent, methylamine and colchicine inhibit the degradation of transplanted enzyme to a much greater extent (85 and 56% respectively). Fluorescence microscopy (with fluorescein isothiocyanate-conjugated mitochondrial outer membrane) shows that transplanted mitochondrial outer membrane undergoes internalization and translocation to a sided perinuclear site, as observed previously with whole mitochondria [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The effects of the inhibitors on the distribution of transplanted membrane material in the cell and inhibition of proteolysis show the importance of cytomorphology for intracellular protein catabolism.

Animals↗
PubMed1984

The inhibition of lysyl oxidase in vivo by isoniazid and its reversal by pyridoxal. Effect on collagen cross-linking in the chick embryo.

Isonicotinic acid hydrazide (isoniazid) causes a large increase in the salt-solubility of collagen when injected into chick embryos; this change is accompanied by the inactivation of lysyl oxidase (EC 1.4.3.13), the enzyme responsible for initiating cross-link formation in collagen and elastin. In addition, isoniazid markedly decreases the liver content of pyridoxal phosphate. The depletion of pyridoxal phosphate takes approx. 6 h, whereas the inhibition of lysyl oxidase and the increase in collagen solubility occur more slowly. A reversal of these effects of isoniazid can be produced by the subsequent injection of a stoichiometric amount of pyridoxal, supporting the role of pyridoxal as a cofactor for lysyl oxidase. Treatment of chick embryos with beta-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, causes an inhibition of the enzyme, which begins to recover within 24 h but which is not affected by the administration of pyridoxal; with isoniazid inhibition, however, lysyl oxidase activity does not show any sign of recovery by 48 h. It is proposed that isoniazid may cause the inhibition of lysyl oxidase by competing for its obligatory cofactor, pyridoxal phosphate. The potential clinical implications in the therapeutic control of fibrosis are briefly discussed.

Amino Acid Oxidoreductases↗
PubMed1984

The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.

Animals↗
PubMed1987

NADPH-binding component of the superoxide-generating oxidase in unstimulated neutrophils and the neutrophils from the patients with chronic granulomatous disease.

The NADPH-binding component of the neutrophil superoxide-generating oxidase was studied in the particulate oxidase fractions obtained from the neutrophils of normal and chronic-granulomatous-disease (CGD) patients. The molecular mass of the NADPH-binding component of the stimulated human neutrophils, which was labelled with the 2',3'-dialdehyde derivative of NADPH and sodium cyanoboro[3H]hydride, was 66 kDa. The 66 kDa component was also labelled in monocytes, but not in red blood cells, platelets and lymphocytes. The particulate oxidase fractions obtained from the patients with CGD had a diminished amount of FAD, whether they contained cytochrome b558 or not. The fractions labelled with the NADPH analogue showed that CGD patients had the NADPH-binding component in the neutrophils. The molecular mass of the component was identical with that of the normal neutrophils. The patients are thought to have an intact NADPH-binding domain of the oxidase in the neutrophils in spite of a diminished amount of FAD in the particulate fractions. The component of the oxidase in the resting neutrophils was also labelled with the analogue. The molecular mass of the component in the resting neutrophils was identical with that of the stimulated neutrophils, and the component was not phosphorylated during the activation process. These results indicate that the NADPH-binding component of the oxidase, which is specific to phagocytes, is present in the resting neutrophils and that the component does not change with respect to molecular mass during the activation process.

Cytochrome b Group↗
PubMed1988

Oxidation of 3-amino-1-phenylprop-1-enes by monoamine oxidase and their use in a continuous assay of the enzyme.

3-Amino-1-phenylprop-1-ene (cinnamylamine) and some derivatives were examined as substrates for monoamine oxidases A and B in mitochondria. All of the amines examined were readily oxidized by monoamine oxidase B but much less readily by monoamine oxidase A. E-Cinnamylamine was found to have Km 0.025 mM and Vmax. 3.9 nmol/min per mg of mitochondrial protein. Corresponding values with monoamine oxidase A were 0.026 mM and 0.85 nmol/min per mg respectively. Despite their different stereochemistry, E- and Z-N-methylcinnamylamines were almost equally effective as substrates for monoamine oxidase B. The characteristic u.v. absorbance and high absorption coefficient of cinnamaldehyde, the product produced by enzymic oxidation of cinnamylamine, is utilized in a sensitive continuous spectrophotometric assay for both enzymes in the rat and for the assay of a purified monoamine oxidase B from bovine liver.

Animals↗
PubMed1989

Activation of NADPH oxidase by purine and pyrimidine nucleotides involves G proteins and is potentiated by chemotactic peptides.

Human neutrophils and HL-60 leukaemic cells possess an NADPH oxidase which catalyses superoxide (O2-) formation and is activated by the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). In dibutyryl cyclic AMP-differentiated HL-60 cells, ATP and UTP in the presence of cytochalasin B activated O2- formation with EC50 values of 5 microM and efficacies amounting to 30% of that of fMet-Leu-Phe. The potency order of purine nucleotides in activating O2- generation was ATP = adenosine 5'-O-(3-thiotriphosphate) greater than ITP greater than dATP = ADP. Pyrimidine nucleotides activated NADPH oxidase in the potency order UTP greater than dUTP greater than CTP = TTP = UDP. Pertussis toxin completely prevented activation of NADPH oxidase by fMet-Leu-Phe and UTP, whereas the effect of ATP was only partially inhibited. ATP and UTP enhanced O2- generation induced by fMet-Leu-Phe by up to 8-fold, and primed the cells to respond to non-stimulatory concentrations of fMet-Leu-Phe. Activation of NADPH oxidase by UTP but not by ATP was inhibited by various activators of adenylate cyclase. In dimethyl sulphoxide-differentiated HL-60 cells and in human neutrophils, ATP and UTP per se did not activate NADPH oxidase, but they potentiated the effect of fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via purino- and novel pyrimidinoceptors respectively, which are coupled to guanine nucleotide-binding proteins leading to the activation of NADPH oxidase. As ATP and UTP are released from cells under physiological and pathological conditions, these nucleotides may play roles as intercellular signal molecules in the activation of O2- formation.

Adenosine Triphosphate↗
PubMed1989

Kinetics of cell-free activation of neutrophil NADPH oxidase. Effects of neomycin and guanine nucleotides.

The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTP gamma S on the kinetics of cell-free activation of NADPH oxidase in membranes of resting human neutrophils were investigated. Arachidonate-mediated activation of the oxidase followed a first-order reaction course (kobs. = 0.39 min-1 at 26 degrees C). In the presence of NaF during the activation process, activity was enhanced while the activation rate was slightly reduced (kobs. = 0.25 min-1 at 26 degrees C). Neomycin blocked activation (half-maximal effect at 25 microM) without affecting rates of superoxide release by preactivated enzyme in vitro or in vivo. In spite of reduced specific activity neither the first-order rate constant of the activation nor the Km of the oxidase were altered by neomycin. Oxidase activated in the presence of GTP gamma S exhibited increased specific activity and unchanged Km; the course of the reaction deviated from first-order kinetics. Kinetic evidence is presented for two separate activation reactions: a GTP gamma S-independent, basal, first-order process and a GTP gamma S-dependent sigmoid activation process. The results are compatible with the existence in neutrophil membranes of two separate pools of dormant oxidase. An alternative scheme of the formation of two active forms of NADPH oxidase is also presented.

Enzyme Activation↗
PubMed1991

Induction of acyl-CoA oxidase and cytochrome P450IVA1 RNA in rat primary hepatocyte culture by peroxisome proliferators.

We have characterized the induction of acyl-CoA oxidase and cytochrome P450IVA1 RNAs in a primary hepatocyte culture system in vitro, using a sensitive and specific RNAse protection assay. Hepatocytes were cultured with a maximal inducing dose of the peroxisome proliferator clofibric acid (1 mM), or vehicle control, for 4 days, and the level of RNAs compared with the level in rats which had been treated with corn oil or clofibric acid (300 mg/kg) for 4 days. The level of acyl-CoA oxidase and P450IVA1 RNAs in 4-day-old control hepatocytes was less than 2% of that in control liver. However, the level of these RNAs in RNA from treated hepatocytes was 61% of that in liver RNA from treated rats. Hepatocytes were treated with the potent peroxisome proliferator methylclofenapate (100 microM), and the induction of RNAs determined at various times after exposure. P450IVA1 RNA was significantly induced 1 h after dosing, rising to 34-fold above control after 8 h, whereas acyl-CoA oxidase RNA was not significantly induced until 4 h, increasing to 5.2-fold above control after 8 h. A similar time course of induction was seen after treatment of hepatocytes with 100 microM-nafenopin, 100 microM-methylclofenapate, 1 mM-clofibric acid or 1 mM-mono(ethylhexyl) phthalate, suggesting that the differential time course of induction of P450IVA1 and acyl-CoA oxidase RNAs is not related to the esterification, structure or potency of the peroxisome proliferator, but is intrinsic to the process of peroxisome proliferation. Hepatocytes were treated with methylclofenapate in the presence and absence of cycloheximide. P450IVA1 RNA was significantly induced by methylclofenapate in the presence of cycloheximide, rising to 17-fold above control after 8 h. However, no induction of acyl-CoA oxidase RNA was detected in the presence of cycloheximide. Therefore we characterize the induction of acyl-CoA oxidase and P450IVA1 RNAs in primary hepatocyte culture in vitro as a faithful model of the induction response in rat liver, and suggest that induction of P450IVA1 RNA is a primary event in the process of peroxisome proliferation.

Acyl-CoA Oxidase↗
PubMed1995

Plasma semicarbazide-sensitive amine oxidase activity is elevated in diabetes mellitus and correlates with glycosylated haemoglobin.

1. Semicarbazide-sensitive amine oxidase is a common name for a group of heterogeneous amine oxidases which are present in various mammalian tissues, especially in vascular smooth muscle cells, cartilage and adipose tissue, but also in plasma. 2. Plasma semicarbazide-sensitive amine oxidase activity was elevated in a group of 104 patients with insulin-dependent diabetes mellitus compared with normal control subjects (555 +/- 172 versus 352 +/- 102 m-units/l, P < 0.0005). 3. Plasma semicarbazide-sensitive amine oxidase activity was higher in subgroups with either retinopathy or nephropathy or both [583 +/- 116 (n = 34), 581 +/- 229 (n = 10) and 646 +/- 249 m-units/l (n = 19), respectively] than in the subgroup without overt complications [486 +/- 129 m-units/l (n = 41), P < 0.005]. 4. Plasma semicarbazide-sensitive amine oxidase activity was positively correlated with plasma glycosylated haemoglobin (r = 0.40; P < 0.0001) and with log urinary albumin excretion (r = 0.26; P < 0.025). 5. The possibility that semicarbazide-sensitive amine oxidase, by its conversion of endogenous amines like methylamine and aminoacetone into cytotoxic aldehydes, plays a role in the development of microvascular complications in diabetes mellitus, needs further investigation.

Adult↗