Xanthine oxidase and aldehyde oxidase: a simple procedure for the simultaneous purification from rat liver

Aldehyde oxidase (AO) and xanthine oxidase (XO) are cytosolic enzymes that have been involved in some pathological conditions and play an important role in the biotransformation of drugs and xenobiotics. The increasing interest in these enzymes demands for a simple and rapid procedure for their purification. This paper describes for the first time a method that allows simultaneous purification of both enzymes from the same batch of rat livers. It involves few steps, is reproducible and offers high enzyme yields with high specific activities. The rat liver homogenate was fractionated by heat denaturation and by ammonium sulphate precipitation to give a crude extract containing both enzymes. This extract was chromatographed on an Hydroxyapatite column that completely separated AO from XO. Further purification of XO by anion exchange chromatography on a Q-Sepharose Fast Flow column resulted in a highly purified (1200-fold) preparation, with a specific activity of 3.64 U/mg and with a 20% yield. AO was purified about 1000-fold at a yield of 15%, with a specific activity of 3.48 U/mg, by affinity chromatography on Benzamidine-Sepharose 6B. The purified enzymes gave single bands of approximately 300 kDa on a polyacrylamide gel gradient electrophoresis and displayed the characteristic absorption spectra of highly purified enzymes.     

Xanthine oxidase activity in regenerating liver

The xanthine oxidase activity of mouse regenerating liver has been shown to be elevated during the period of rapid liver growth and proliferation. This increase is evident when the enzyme activity is expressed per unit wet tissue weight, per unit nitrogen, or per cell. The adrenal cortex probably plays only a minor role in implementing this phenomenon. Further augmentation of the xanthine oxidase level of regenerating liver is not induced by the administration of large quantities of the substrate, xanthine, to the animal.     

Immunoaffinity purification and characterization of leucine aminopeptidase from human liver

Leucine aminopeptidase was purified from human liver cytosol to homogeneity, 1538-fold, with a yield of 84.4% by immunoaffinity chromatography. Increases in the activity and the stability of the enzyme were simultaneously observed during the purification procedure, suggesting the presence of some endogenous inhibitor in cytosol. The specific activity and Km value of the enzyme for L-leucine amide were found to be 58.00 mumol/min/mg of protein and 4.02 mM, respectively, at pH 8.0. The molecular weight of the enzyme was determined to be 360,000 by both polyacrylamide gradient gel electrophoresis and Sephadex G-200 gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of native and dimethyl suberimidate cross-linked enzyme indicate that the native enzyme has two subunits of Mr 53,000 (a) and 65,000 (b) and is a hexamer arranged as a trimer of dimers (3 X (a X b)). The optimum pH was 10.5, and the enzyme was stable in the pH range from 7.5-8.5. The enzyme was activated by divalent metal ions, especially by Mg2+ and Mn2+, with no change in Km value. The enzyme was inhibited by metal-chelating agents, indicating it to be a metalloenzyme. Amastatin and bestatin strongly inhibited the enzyme, but leupeptin did not. The enzyme had a broad substrate specificity toward oligopeptides and amino acid amides but had little or no activity toward chromogenic substrates. The enzyme also could hydrolyze natural substrates contained in liver cytosol and accordingly produce many kinds of amino acids commonly found in proteins.     

Quinolinic acid phosphoribosyltransferase: purification and partial characterization from human liver and brain

Quinolinic acid phosphoribosyltransferase (QPRT) [EC 2.4.2.19] from human liver and brain was purified to homogeneity. Identity of the pure enzymes isolated from the two organs was proven by biochemical, physiocochemical and, following the production and partial purification of anti-liver QPRT antibodies, immunological techniques. Human QPRT has a molecular weight of 170,000 and consists of five identical subunits. Kinetic analyses revealed a Km of 5.6 microM for the substrate (quinolinic acid) and 23 microM for the co-substrate (phosphoribosylpyrophosphate). Enzyme activity was dependent on Mg2+ (optimal concentration: 1 mM) and was inhibited by the enzymatic by-product, inorganic pyrophosphate. Pure QPRT and its antibodies will constitute useful tools in the examination of the possible role of quinolinic acid in the pathogenesis of human neurodegenerative disorders.     

Isolation and characterization of glycolic acid oxidase from human liver.

Glycolic acid oxidase has been isolated from human liver and purified over 3000-fold to a specific activity of 123 U/mg protein by a 5-step procedure. The preparation gave a single protein band on polyacrylamide gel electrophoresis, required flavin mononucleotide for catalytic activity, had a pH optimum between 8.2-8.8 depending on the substrate, and had a molecular weight of 105 000. The enzyme has a broad specificity towards alpha-hydroxy acids. Glycolate (Km = 3.3 . 10(-4) M) was the most effective substrate. The enzyme was stable for several months when stored as an (NH4)2SO4 precipitate or in 15% glycerol. Since glycolate inhibits the oxidation of glyoxylate to oxalate by glycolic acid oxidase, it is suggested that glycolic acid oxidase contributes to the synthesis of oxalate in vivo when the glyoxylate concentration is high and the glycolate concentration is low.     

Xanthine oxidoreductase and xanthine oxidase in human cornea

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.     

Guinea pig liver aldehyde oxidase as a sulfoxide reductase: Its purification and characterization

Guinea pig aldehyde oxidase was purified about 120-fold at a yield of 26% from liver cytosol by sequential column chromatography using DEAE-cellulose, FMN-Sepharose 4B, and Sephacryl S-300. The purified enzyme showed many similarities with the rabbit liver aldehyde oxidase reported by other workers with respect to its absolute spectra, molecular weight, and cofactor compositions of molybdenum, FAD, and nonheme iron. This enzyme efficiently utilized 2-hydroxypyrimidine and benzaldehyde as electron donors while N¹-methylnicotinamide was 40 times less effective than 2-hydroxypyrimidine. Diphenyl sulfoxide was reduced anaerobically to diphenyl sulfide in the presence of electron donors. This activity was highly susceptible to SKF 525-A as well as the known inhibitors for aldehyde oxidase such as menadione, estradiol, and potassium cyanide. This enzyme also reduced dibenzyl sulfoxide, phenothiazine sulfoxide, d-biotin methyl ester d-sulfoxide, and quinoline N-oxide, but not l-methionine sulfoxide, dimethyl sulfoxide, d-biotin methyl ester l-sulfoxide, and d-biotin d- and l-sulfoxides, as well as diphenyl sulfone. These results indicate that aldehyde oxidase in guinea pig liver functions as a sulfoxide reductase with selective substrate specificity under anaerobic conditions.     

Purification and immunochemical characterization of monoamine oxidase from rat and human liver.

1. Monoamine oxidase from rat and human liver was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. The enzyme activity was extracted from mitochondrial preparations by Triton X-100. The enzyme was purified by (NH4)2SO4 fractionation followed by chromatography on DEAE-cellulose, Sepharose 6B, spheroidal hydroxyapatite, and finally chromatography on diazo-coupled tyramine-Sepharose. 3. Distinct differences occur in the chromatographic behaviour of the two enzymes on both DEAE-cellulose and spheroidal hydroxyapatite. 4. It is unlikely that the purification of the enzymes on tyramine-Sepharose is due to affinity chromatography and reasons for this are discussed. 5. The purified enzymes did not oxidize-5-hydroxytryptamine and the relative activities of the enzymes with benzylamine were increased approx. 1.25-fold compared with the enzyme activities of mitochondrial preparations. 6. Immunotitration of enzyme activity in extracts of mitochondrial preparations from rat liver was carried out with 5-hydroxytryptamine, tyramine and benzylamine. The enzyme activities were completely immunoprecipitated by the same volume of antiserum. Similar results were obtained with the antiserum to the enzyme from human liver.     

Purification and characterization of recombinant human liver glycolate oxidase

Glycolate oxidase, an FMN-dependent peroxisomal oxidase, plays an important role in plants, related to photorespiration, and in animals, where it can contribute to the production of oxalate with formation of kidney stones. The best studied plant glycolate oxidase is that of spinach; it has been expressed as a recombinant enzyme, and its crystal structure is known. With respect to animals, the enzyme purified from pig liver has been characterized in detail in terms of activity and inhibition, the enzyme from human liver in less detail. We describe here the purification and initial characterization of the recombinant human glycolate oxidase. Its substrate specificity and the inhibitory effects of a number of anions are in agreement with the properties expected from previous work on glycolate oxidases from diverse sources. The recombinant enzyme presents an inhibition by excess glycolate and by excess DCIP, which has not been documented before. These inhibitions suggest that glycolate binds to the active site of the reduced enzyme, and that DCIP also has affinity for the oxidized enzyme. Glycolate oxidase belongs to a family of l-2-hydroxy-acid-oxidizing flavoenzymes, with strongly conserved active-site residues. A comparison of some of the present results with studies dealing with other family members suggests that residues outside the active site influence the binding of a number of ligands, in particular sulfite.     

Xanthine oxidase inhibitors isolated from Piper nudibaccatum

Two new hydroxychavicol analogs nudibaccatumin A (1) and B (2), together with twenty known compounds were isolated from the methanol extract of Piper nudibaccatum. Their structures were elucidated by extensive spectroscopic analyses (1D and 2D NMR, HRESIMS, UV, IR and polarimetry). Hydroxychavicol is a known inhibitor of xanthine oxidase (XO). In the present study, hydroxychavicol and 5 natural analogs (1–5) were evaluated for their XO inhibitory activity. Neotaiwanensol B (3) (IC₅₀ = 0.28 μM) showed a greater inhibitory effect than hydroxychavicol and allopurinol (the positive control). Two new compounds 1 and 2 showed a moderate inhibition activity with an IC₅₀ value of 62.94 μM and 70.67 μM, respectively.     

Nicotinamide riboside phosphorylase from beef liver: purification and characterization

Nicotinamide riboside phosphorylase (NR phosphorylase) from beef liver has been purified to apparent homogeneity at 300-fold purification with a 35% yield. Kinetic constants for the enzyme-catalyzed phosphorolysis were as follows Knicotinamide riboside, 2.5 +/- 0.4 mM; Kinorganic phosphate, 0.50 +/- 0.12 mM; Vmax, 410 +/- 30 X 10(-6) mol min-1 mg protein-1, respectively. The molecular weights of the native enzyme and subunit structure were determined to be 131,000 and 32,000, respectively, suggesting the beef liver NR phosphorylase to be tetrameric in structure and consistent with the presence of identical subunits. The amino acid composition was shown to be very similar to that reported for human erythrocyte purine-nucleoside phosphorylase but differing considerably from that found for rat liver purine-nucleoside phosphorylase. In addition to catalytic activity with nicotinamide riboside, the beef liver enzyme catalyzed a phosphorolytic reaction with inosine and guanosine exhibiting activity ratios, nicotinamide riboside:inosine: guanosine of 1.00:0.35:0.29, respectively. These ratios of activity remained constant throughout purification of the beef liver enzyme and no separation of these activities was detected. Phosphorolysis of nicotinamide riboside was inhibited competitively by inosine (Ki = 75 microM) and guanosine (Ki = 75 microM). Identical rates of thermal denaturation of the beef liver enzyme were observed when determined for the phosphorolysis of either nicotinamide riboside or inosine. These observations coupled with studies of pH and specific buffer effects indicate the phosphorolysis of nicotinamide riboside, inosine, and guanosine to be catalyzed by the same enzyme.     

The purification and characterization of glutaryl-coenzyme A dehydrogenase from porcine and human liver

Glutaryl-CoA dehydrogenase, a multifunctional enzyme responsible for dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA, has been purified 1,680-fold from porcine liver mitochondria. The purified porcine enzyme has a subunit molecular weight of 47,800 and a native molecular weight of 190,500. Porcine glutaryl-CoA dehydrogenase catalyzed the conversion of [1,5-14C]glutaryl-CoA to [14C] crotonyl-CoA and 14CO2 in a 1:1:1 ratio. The porcine enzyme has Km values for electron transfer flavoprotein and glutaryl-CoA of 1.1 and 3.3 microM, respectively, and turnover numbers of 860 mol of electron transfer flavoprotein/min/mol of glutaryl-CoA dehydrogenase and 327 mol of glutaryl-CoA/min/mol of glutaryl-CoA dehydrogenase. Human glutaryl-CoA dehydrogenase has been purified 1,278-fold from human liver mitochondria. The purified human enzyme has a subunit molecular weight of 58,800 and a native molecular weight of 256,000. Human glutaryl-CoA dehydrogenase showed a reaction of only partial identity when compared to porcine glutaryl-CoA dehydrogenase by Ouchterlony double immunodiffusion analysis using antiserum raised against and monospecific for porcine glutaryl-CoA dehydrogenase.     

Acid sphingomyelinase from human urine: purification and characterization

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine in the presence of 0.1% Nonidet P-40. The activity could be enriched 23,000-fold by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose and DEAE-cellulose. The last purification step yielded an enzyme preparation with a specific activity of about 2.5 mmol sphingomyelin cleaved/h per mg protein and with a yield of about 3%. Purified sphingomyelinase appeared to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 70 kDa. In the presence of 0.08% (w/v) sodium taurodeoxycholate the preparation showed phosphodiesterase activity toward sphingomyelin, phosphatidylcholine and phosphatidylglycerol. These activities co-purified during the entire purification procedure, indicating that the acid sphingomyelinase hydrolyses not only sphingomyelin but also the other two phospholipids, phosphatidylcholine and phosphatidylglycerol. Addition of 100 microM tripalmitoylglycerol to the assay system (which contains 100 microM sphingomyelin) instead of detergent, stimulated the reaction about 20-fold compared to an assay which did not contain detergents, thus offering a very sensitive and efficient system for the assay of sphingomyelinase in a system free of detergents. Sphingomyelin degradation was strongly inhibited by phosphatidylinositol 4',5'-bisphosphate, adenosine 3',5'-diphosphate and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (50% inhibition at inhibitor concentrations of 1-5 microM).     

Partial purification and characterization of thiosulfate oxidase from Pseudomonas aeruginosa.

Soluble thiosulfate oxidase from Pseudomonas aeruginosa was purified 85-fold and coverted thiosulfate to tetrathionate by using either ferricyanide or cytochrome c as an electron acceptor.