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PubMedDate not supplied

Quantitation of multiple pathways for the metabolism of nephrotoxic cysteine conjugates using selective inhibitors of L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and cysteine conjugate beta-lyase.

In this study, we have established the selectivity of inhibitors for rat kidney cysteine conjugate beta-lyase and L-alpha-hydroxy acid oxidase (L-amino acid oxidase) and have used these inhibitors to explore the relative roles of these two enzymes in the metabolism of nephrotoxic cysteine conjugates by rat kidney homogenate. In addition, we have investigated the relationship between structure and the metabolism of toxic cysteine conjugates by purified rat kidney L-alpha-hydroxy acid oxidase. With purified enzyme, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBDC) was about four times more active than S(1,2-dichlorovinyl)-L-cysteine (DCVC). Three alkyl conjugates were less active than DCVC. Purified L-alpha-hydroxy acid oxidase was not inhibited by the beta-lyase inhibitor aminooxyacetic acid but was inactivated by 2-hydroxy-3-butynoate. PCBDC metabolism in rat kidney homogenate was inhibited 74% by aminooxyacetic acid and 42% by 2-hydroxy-3-butynoate, whereas DCVC metabolism was inhibited 77% by aminooxyacetic acid and 28% by 2-hydroxy-3-butynoate. However, only aminooxyacetic acid inhibited the binding of 35S label from [35S]DCVC. Based on these results we have reached three conclusions. First, L-alpha-hydroxy acid oxidase plays a significant role in the metabolism of some cysteine conjugates. Second, metabolism of DCVC by L-alpha-hydroxy acid oxidase does not contribute directly to covalent binding. Third, as much as 65% of DCVC may be metabolized to its corresponding alpha-keto acid. The results are discussed with regard to the nephrotoxicity of cysteine conjugates.

Amino Acid Oxidoreductases↗
PubMed1983

Measurement of superoxide dismutase, diamine oxidase and caeruloplasmin oxidase in the blood of thoroughbreds.

Methods were developed for the measurement of superoxide dismutase (SOD), diamine oxidase (DAO) and caeruloplasmin oxidase in the blood of thoroughbred horses. These enzymes were measured in 178 normal thoroughbreds stabled throughout the United Kingdom. The relationships between the activities of SOD, DAO and caeruloplasmin oxidase and the blood concentrations of their associated trace metals (copper, zinc and manganese) were studied in 52 of the thoroughbreds. Trace metals were measured by electrothermal atomic absorption spectrophotometry. No relationships were found between the activities of erythrocyte SOD and serum/whole blood copper, zinc and manganese, or serum DAO and serum copper or zinc concentrations. Caeruloplasmin oxidase in equine blood was found to be correlated to serum copper concentration, r = 0.695 (P less than 0.001) over the normal range. Samples from thoroughbreds with trace metal deficiency or toxicity were not available for study. The observed normal ranges for the activity of these enzymes are as follows: SOD: 50 to 200 units per ml whole blood between 5 and 95 percentiles; DAO: 0.1 to 28.5 units per litre (means = 14.8, SD 7.1) and caeruloplasmin oxidase; 11.6 to 35.8 units per ml (means = 23.7, SD 6.0). For numerical simplicity, the activity of DAO is given in units per litre, compared to units per ml for caeruloplasmin oxidase and SOD.

Amine Oxidase (Copper-Containing)↗
PubMed1993

Translocation of Rac correlates with NADPH oxidase activation. Evidence for equimolar translocation of oxidase components.

Activation of the superoxide-generating NADPH oxidase system of human neutrophils involves the assembly of several neutrophil components, some located on the plasma membrane and others in the cytosol. It has recently been established that one of the required components for NADPH oxidase activity is the GTP-binding protein Rac. To further investigate the role of Rac in the NADPH oxidase system, studies were carried out to determine its subcellular distribution in resting and activated human neutrophils. In resting cells, Rac and an associated guanine nucleotide regulatory factor, GDP dissociation inhibitor (GDI), were located only in the cytosol, along with other known oxidase factors, p47-phox and p67-phox. After activation of neutrophils with phorbol 12-myristate 13-acetate or formyl-methionyl-leucyl-phenylalanine, Rac was translocated from the cytosol to the plasma membrane, and this translocation corresponded temporally with the translocation of p47-phox and p67-phox and with the generation of superoxide. GDI remained localized to the cytosol, suggesting activation of the oxidase involved dissociation of the Rac-GDI complex prior to Rac translocation. Determination of the quantities of cytosolic factors associated with the plasma membrane indicated that Rac, p47-phox, and p67-phox are translocated to the plasma membrane simultaneously in equimolar amounts, but that the membrane-associated cytochrome b was present at 3-4-fold molar excess. These findings suggest that Rac may play a role in assembly of the active NADPH oxidase complex.

Biological Transport↗
PubMed1993

The reaction of Bacillus subtilis aa3-600 oxidase with oxygen: an aa3-oxidase lacking the CuA site.

The reaction of reduced cytochrome aa3-600 nm oxidase from Bacillus subtilis with oxygen has been measured. The reaction of fully reduced oxidase with oxygen was studied by the flow-flash method which employs the photosensitive CO complex to protect the enzyme from oxygen until the reaction is initiated by a photolytic flash. The rate of the reaction, measured in the Soret region, is oxygen concentration dependent at low levels of oxygen and becomes independent from oxygen above 200 microM. The reaction reaches a rate limit of 9500 s-1 at high oxygen. This type of oxygen concentration dependence indicates that the oxidation reaction is preceded by another event, probably an oxygen combination reaction. When compared to mammalian cytochrome oxidase the Bacillus oxidase exhibits a simpler reaction profile. This simpler observed reaction profile can be accounted for by the absence of CuA in the Bacillus oxidase using the same model developed for the single turnover of mammalian cytochrome oxidase.

Bacillus subtilis↗
PubMed1984

Immunological similarity of milk sulfhydryl oxidase and kidney glutathione oxidase.

A radioimmunoassay for sulfhydryl oxidase, a membrane enzyme, was developed using antibodies raised to the bovine milk enzyme which had been purified by transient covalent affinity chromatography on a cysteinylsuccinamidopropyl-glass matrix. Bovine milk sulfhydryl oxidase and bovine kidney sulfhydryl oxidase ("glutathione oxidase") appear to be immunologically identical as evidenced by parallel responses in radioimmunoassays. Antibodies raised to the purified milk sulfhydryl oxidase can immunoprecipitate glutathione oxidase activity, but not gamma-glutamyltransferase ("transpeptidase") activity, from bovine kidney preparations.

Animals↗
PubMed1999

Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus.

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. We previously identified and purified an alternative oxidase, cytochrome bd-type quinol oxidase, from a mutant of Bacillus stearothermophilus defective in the caa3-type oxidase activity (J. Sakamoto et al., FEMS Microbiol. Lett. 143 (1996) 151-158). Compared with proteobacterial counterparts, B. stearothermophilus cytochrome bd showed lower molecular weights of the two subunits, shorter wavelength of alpha-band absorption maximum due to heme D, and lower quinol oxidase activity. Preincubation with menaquinone-2 enhanced the enzyme activity up to 40 times, suggesting that, besides the catalytic site, there is another quinone-binding site which largely affects the enzyme activity. In order to clarify the molecular basis of the differences of cytochromes bd between B. stearothermophilus and proteobacteria, the genes encoding for the B. stearothermophilus bd was cloned based on its partial peptide sequences. The gene for subunit I (cbdA) encodes 448 amino acid residues with a molecular weight of 50195 Da, which is 14 and 17% shorter than those of Escherichia coli and Azotobacter vinelandii, respectively, and CbdA lacks the C-terminal half of the long hydrophilic loop between the putative transmembrane segments V and VI (Q loop), which has been suggested to include the substrate quinone-binding site for the E. coli enzyme. The gene for subunit II (cbdB) encodes 342 residues with a molecular weight of 38992 Da. Homology search indicated that the B. stearothermophilus cbdAB has the highest sequence similarity to ythAB in B. subtilis genome rather than to cydAB, the first set of cytochrome bd genes identified in the genome. Sequence comparison of cytochromes bd and their homologs from various organisms demonstrates that the proteins can be classified into two subfamilies, a proteobacterial type including E. coli bd and a more widely distributed type including the B. stearothermophilus enzyme, suggesting that the latter type is evolutionarily older.

Amino Acid Sequence↗
PubMed1998

Human sulfite oxidase R160Q: identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme.

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in Km for sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km. Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.

Child, Preschool↗
PubMed2006

A tissue-specific variant of the human lysyl oxidase-like protein 3 (LOXL3) functions as an amine oxidase with substrate specificity.

The human lysyl oxidase-like 3 (LOXL3) encodes a member of the emerging family of lysyl oxidase (LOX) that functions as a copper-dependent amine oxidase. The LOXL3 protein contains four scavenger receptor cysteine-rich domains in the N terminus in addition to the C-terminal characteristic domains of the LOX family, such as a copper binding domain, a cytokine receptor-like domain and residues for the lysyl-tyrosyl quinone cofactor. Using BLASTN searches, we identified a LOXL3 variant LOXL3-sv1 that lacked the sequences corresponding to exons 1, 2, 3, and 5 of LOXL3. LOXL3-sv1 showed an exon-intron structure distinct from LOXL3, additionally containing an 80-bp sequence corresponding to intron 3 of LOXL3 in the 5'-UTR and a 561-bp sequence corresponding to the 3'-flanking genomic region of exon 14 in the 3'-UTR. LOXL3-sv1 was predicted to encode a polypeptide of 392 amino acids that contains the C-terminal domains required for amine oxidase activity but lacks the N-terminal SRCR domains 1, 2, and 3. The recombinant LOXL3-sv1 protein showed a beta-aminopropionitrile-inhibitable amine oxidase activity toward elastin and collagen with substrate specificity. In RT-PCR assays with various human tissues, LOXL3-sv1 and LOXL3 showed distinct expression patterns. Further, luciferase reporter assays revealed a strong promoter element in intron 3 that probably functions as a regulatory region for the expression of LOXL3-sv1. These findings strongly indicate that LOXL3 encodes two variants, LOXL3 and LOXL3-sv1, both of which function as amine oxidases with distinct tissue and substrate specificities from one another.

Amino Acid Oxidoreductases↗
PubMed1983

Mechanism of the increase in cytochrome c oxidase activity in pea cotyledons during seed hydration. The presence of free cytochrome-c-oxidase subunits in dry cotyledons and their probable assembly into the holoenzyme during seed hydration.

The increase in cytochrome c oxidase activity in pea cotyledons during seed hydration was investigated with two kinds of antibodies against the enzymes purified from mitochondria of pea shoot and sweet potato root. The antibody against the pea enzyme recognized only the largest of the enzyme subunits (subunit I), while that against the sweet potato one did other three of the pea enzyme subunits: the second largest (subunit II) and the two smallest (subunits IV and V). There was no increase in the amount of protein immunoprecipitated with the anti-(pea enzyme) antibody in a crude fraction of cotyledon membranes during seed hydration, despite the marked increase in cytochrome c oxidase activity. Neither cycloheximide nor chloramphenicol had any effect on the activity or the amount. The crude membrane fraction from dry cotyledons contained an enzymatically inert protein precipitated with the anti-(pea enzyme) antibody. The inert protein could be separated from the active enzyme protein through solubilization of the protein from the membranes with various concentrations of Triton X-100. It consisted of only one kind of polypeptide with the same molecular weight as subunit I and decreased with the concomitant increase in the active enzyme protein during seed hydration. Dry pea cotyledons also contained a soluble protein that was immunoprecipitated by antibody to the sweet potato enzyme. The soluble form of the immunoreactive protein decreased with the concomitant increase in active cytochrome c oxidase protein in the crude membrane fraction from the cotyledons during seed hydration. None of these changes during seed hydration were inhibited by cycloheximide or chloramphenicol. The soluble, immunoreactive protein contained only one kind of polypeptide, of which the molecular weight was identical to that of subunit IV. The mitochondrial inner membranes of dry cotyledons were separated into three fractions by sucrose density gradient centrifugation. The lightest was the richest of the three in the enzymatically inert protein immunoprecipitated with the anti-(pea enzyme)antibody; cytochrome c oxidase activity in this fraction increased immediately after the onset of seed hydration. These results indicate that a membrane-bound, free form of subunit I and a soluble, free form of subunit IV of cytochrome c oxidase exist in dry pea cotyledons. We propose that these free forms of subunits are assembled with the other subunits to form the active cytochrome c oxidase during seed hydration.

Electron Transport Complex IV↗
PubMed1994

The heme-copper oxidase family consists of three distinct types of terminal oxidases and is related to nitric oxide reductase.

Among aerobic prokaryotes, many different terminal oxidase complexes have been described. Sequence comparison has revealed that the aa3-type cytochrome c oxidase and the bo3-type quinol oxidase are variations on the same theme: the heme-copper oxidase. A third member of this family has recently been recognized: the cbb3-type cytochrome c oxidase. Here we give an overview, and report that nitric oxide (NO) reductase, a bc-type cytochrome involved in denitrification, shares important features with these terminal oxidases as well. Tentative structural, functional and evolutionary implications are discussed.

Amino Acid Sequence↗
PubMed2006

Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans.

It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

Acidithiobacillus thiooxidans↗
PubMed1975

Covalently bound FAD in d-6-hydroxynicotine oxidase. Immunological studies of D- and L-6-hydroxynicotine oxidase: evidence for a D-enzyme precursor.

Antersera prepared against both enantiozymes, D- and L-6-hydroxynicotine oxidase, formed precipitins in double diffusion tests with their respective antigens only. A mixture of the two antisera caused spur formation of the two precipitin lines obtained with the pure enzymes. Antiserum to L-apoprotein reacted with native L-enzyme and L-apoprotein but not with the D-sspecific enzyme. D-6-hydroxynicotine oxidase activity was inhibited by the anti-D-antiserum, leaving the L-enzyme fully active, while anti-L-antiserum inhibited the L- but not the D-specific activity. The delayed induction of D-6-hydroxynicotine oxidase as compared to the other activities of the nicotine-degrading sequence and the differential immunochemical behavior of the enantiozymes allowed the search for a D-enzyme precursor. In cells harvested 3 hours after the addition of DL-nicotine, the L-enzyme activity was present, whereas no D-enzyme activity could be detected. However, an extract of these cells did form an immunoprecipitin line with anti-D-antiserum. L-6-Hydroxynicotine oxidase, but no D-6-hydroxynicotine oxidase activity, could also be induced in Arthrobacter oxidans grown in a medium with a high glucose content and DL-nicotine as the sole nitrogen source. An extract of these L-induced cells produced the specific immunoprecipitation with anti-D-antiserum. A pulse-chase experiment with cells grown first on glucose and DL-nicotine in the presence of [14C]leucine and then in an unlabeled medium which induces D-6-hydroxynicotine oxidase activity resulted in a radioactive D-enzyme-immunoprecipitin line. From these experiments it is concluded that a precursor of the active D-enzyme is induced simultaneously with the other nicotine-degrading enzymes.

Arthrobacter↗
PubMedDate not supplied

Tissue distribution of the molybdenum hydroxylases, aldehyde oxidase and xanthine oxidase, in male and female guinea pigs.

The activity of the molybdenum hydroxylase, aldehyde oxidase, was determined in crude homogenates and (NH4)2SO4 fractions prepared from guinea pig liver, lung, kidney, intestine, spleen and heart. Xanthine oxidase was also measured in (NH4)2SO4 fractions. In each case, xanthine oxidase levels were lower than those of aldehyde oxidase; activity of the latter enzyme was highest in the liver, whereas xanthine oxidase was predominant in the small intestine. There was no significant difference in the activity of either molybdenum hydroxylase between tissues taken from male and female guinea pigs.

Aldehyde Oxidase↗
PubMed2003

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies.

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3' of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu2+. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by beta-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.

Amine Oxidase (Copper-Containing)↗
PubMed2002

Molecular analysis of the bison phagocyte NADPH oxidase: cloning and sequencing of five NADPH oxidase cDNAs.

During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of a superoxide anion-generating complex known as the NADPH oxidase. Despite the importance of this system in animal host defense, almost nothing is known about the NADPH oxidase in neutrophils from wild ruminant species. In the present studies, we provide a molecular analysis of the bison leukocyte NADPH oxidase. Using reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends, we cloned and sequenced the full-length cDNAs for five bison NADPH oxidase components: p22(phox), p40(phox), p47(phox) and p67(phox), and gp91(phox). When compared to other species, the deduced amino acid sequences of the bison homologs were most similar to those of bovine. Interestingly, a bison p40(phox) alternative splice product was isolated, which was similar to that observed for human p40(phox) in that the cDNAs contained sequence from intron 8. Consistent with the high degree of similarity between bison and bovine amino acid sequences, immunoblot analysis showed that the bison homologs migrated similarly to their bovine counterparts. Overall, these studies show that the bison and bovine NADPH oxidase genes are highly conserved between these two species, despite their divergence from a common ancestor over 1 million years ago.

Alternative Splicing↗