Takeshi Nishino

The conversion from the dehydrogenase type to the oxidase type of rat liver xanthine dehydrogenase by modification of cysteine residues with fluorodinitrobenzene.

When rat liver xanthine dehydrogenase was incubated with fluorodinitrobenzene (FDNB) at pH 8.5, the total enzyme activity decreased gradually to a limited value of initial activity with modification of two lysine residues in a similar way to the modification of bovine milk xanthine oxidase with FDNB (Nishino, T., Tsushima, K., Hille, R. and Massey, V. (1982)J. Biol. Chem. 257, 7348–7353). After modification with FDNB, the two peptides containing dinitrophenyl-lysine were isolated from the molybdopterin domain after proteolytic digestion and were identified as Lys754 and Lys771 by sequencing the peptides. During the modification of these lysine residues, xanthine dehydrogenase was found to be converted to an oxidase form in the early stage of incubation. Incorporation of the3H-dinitrophenyl group into enzyme cysteine residues was 0.96 mol per enzyme FAD for 68% conversion to the oxidase form. The modified enzyme was reconverted to the dehydrogenase form by incubation with dithiothreitol with concomitant release of3H-dinitrophenyl compounds. After modification with3H-FDNB followed by carboxymethylation under denaturating conditions, the enzyme was digested with proteases. Three3H-dinitrophenyl-labeled peptides were isolated and sequenced. The modified residues were identified to be Cys535, Cys992 and Cys1324. These residues are conserved among the all known mammalian enzymes, but Cys992 and Cys1324 are not conserved in the chicken enzyme. Cys1324 of the rat enzyme was found not to be involved in the conversion from the dehydrogenase to the oxidase by limited proteolysis experiments, but Cys535 and Cys992 which seemed to be modified alternatively with FDNB appear to be involved in the conversion.

PURIFICATION OF HIGHLY ACTIVE MILK XANTHINE OXIDASE BY AFFINITY CHROMATOGRAPHY ON SEPHAROSE 4B/FOLATE GEL

Takeshi NISHINO, Tomoko NISHINO and Keizo TSUSHIMA Department of Biochemistry, Yokohama City University School of Medicine, Urafune-cho, Minami-ku, Yokohama 232, Japan Received 9 July 1981 1. Introduction It is well known that an inactive form of xanthine oxidase, the desulfo-form, is present in milk xanthine oxidase preparations, in addition to an inactive form lacking molybdenum [1-4]. The existence of the desulfo-form in the enzyme preparations is considered to be caused by spontaneous release of sulfur during storage or purification. This inactive form is also known to be caused by treatment of the enzyme with cyanide [5]. To understand the proper mechanism of action of this enzyme, it is important to obtain an enzyme preparation essentially free of inactive forms. An affinity chromatography method for resolution of the active and inactive forms of xanthine oxidase was reported in [6]. An allopurinol analogue was used as an affinity ligand [6]. In [6], only the active enzyme, in a reduced form, was bound to the column and could be eluted after reoxidation. Despite the extreme value of affinity chromatography, prepara- tion of the enzyme by their method is not easy, because it involves difficult chemical syntheses and chromatography under anaerobic conditions. Here, we describe a much easier method for puri- fication of highly active enzyme by affinity chroma- tography, in which resolution of the sulfo- and desulfo-enzymes is achieved. We adopted folate [7], a commercially available competitive inhibitor, as a ligand instead of the allopurinol analogue. 2. Materials and methods Xanthine oxidase was prepared from cows milk Abbreviations: DMF, dimethylformamide; EDC, N-ethyl-N- 3 dimethylaminopropyl carbodiimide; AFR, activity flavin ratio by the method in [8] with minor modifications. Cysteine (5 mM), salicylate (1 mM) and EDTA (0.2 mM) were added to the extraction buffer of Na2HPO4 (0.2 M). Final ammonium sulfate fractionation was performed between 30-45% saturation instead of 33-42% saturation as in the original method. 2.1. Preparation of Sepharose 4B/folate gel AH-Sepharose (10 g) obtained from Pharmacia, was swollen in 0.5 M NaC1 and washed with 1 titer of 0.5 M NaC1. Washed AH-Sepharose was mixed with 60 ml 2 mM folate in 50% DMF, the pH of which had been pre-adjusted to 5.8 with dilute NaOH. Then 400 mg EDC, obtained from Fluka, was added and the mixture was gently stirred overnight at room temperature in the dark. The gel was washed sequen- tially with 200 ml of 50% DMF at pH 7.0,500 ml 0.01 M NaOH, 500 ml 0.1 M Tris-HC1 at pH 7.0, and finally with 1 liter of distilled water. The Sepha- rose 4B/folate gel was stored at 4°C in the dark. Oxipurinol was synthesized as in [9]. Allopurinol was obtained from Sigma. Enzyme activity was measured spectrophoto- metrically at 295 nm and at 25°C [4] using a Shimazu 140 spectrophotometer. Catalytic activity was expressed as AFR 25°c [10]. The following buffer mixtures were used for affinity chromatography: (A) Mixture of 20% 0.1 M pyrophosphate buffer (pH 8.5) containing 0.2 mM EDTA and 80% 0.05 M Tris-HC1 buffer (pH 7.8) containing 0.2 mM EDTA. (B) Mixture of 30% of 0.1 M pyrophosphate buffer (pH 8.5) containing 0.2 mM EDTA and 70% 0.05 M Tris-HC1 buffer (pH 7.8) containing 0.2 mM EDTA. Published by Elsevier/North-Holland Biomedical Press 00145793[81[0000-0000/

The presence of desulfo xanthine dehydrogenase in purified and crude enzyme preparations from rat liver

02.50

The mechanism of conversion of xanthine dehydrogenase to oxidase and the role of the enzyme in reperfusion injury.

Crude and purified xanthine dehydrogenase preparations from rat liver were examined for the existence of a naturally occurring inactive form. Reduction of the purified enzyme by xanthine under anaerobic conditions proceeded in two phases. The enzyme was inactivated by cyanide, which caused the release of a sulfur atom from the molybdenum center as thiocyanate. The amount of thiocyanate released was almost in parallel with the initial specific activity. The active and inactive enzymes could be resolved by affinity chromatography on Sepharose 4B/folate gel. These results provided evidence that the purified enzyme preparation from rat liver contained an inactive form. A method for the determination of the active and inactive enzymes in crude enzyme preparations from rat liver was devised based on the fact that only active enzyme could react with [14C]allopurinol and both active and inactive enzymes could be immunoprecipitated quantitatively by excess specific antibody to xanthine dehydrogenase. The amount of [14C]alloxanthine (derived from [14C]allopurinol) bound to the active sulfo enzyme in crude rat liver extracts was about 0.5 mol/mol of FAD. As this content is closely similar to that in the purified enzyme, these results suggest the existence of an inactive desulfo form in vivo.

A Novel Xanthine Dehydrogenase Inhibitor (BOF-4272)

In 1968 Delia Corte and Stirpe reported that mammalian xanthine oxidase was originally a dehydrogenase, and was converted to the oxidase by modification of the enzyme protein during extraction or purification (Delia Corte and Stirpe, 1968, 1972). This conversion occurs either reversibly by sulfhydryl oxidation, or irreversibly by proteolysis (Delia Corte and Stirpe, 1968, 1972; Stirpe and Delia Corte, 1969; Waud and Rajagopalan,1976; Nakamura and Yamazaki, 1982; Ikegami and Nishino, 1986; Saito and Nishino, 1989). Although the mammalian enzyme is easily converted from the dehydrogenase to the oxidase, the enzyme can be purified in a reversible form by careful purification procedures (Waud and Rajagopalan,1976; Nakamura and Yamazaki, 1982; Ikegami and Nishino, 1986; Saito and Nishino, 1989). The dehydrogenase form has very low oxidase activity (3–4% of dehydrogenase activity) when enzyme activity is determined by the standard assay system, but detailed kinetic analysis shows that even the dehydrogenase has an appreciable amount of oxidase activity. Table 1 shows the kinetic parameters obtained from rat liver xanthine dehydrogenase and oxidase (Saito and Nishino, 1989). For the dehydrogenase, the Vmax value for dehydrogenase activity has about four times higher than the value for oxidase activity. The Km value for oxygen of the dehydrogenase is five times higher than that of the oxidase. The oxidase activity of the dehydrogenase is almost completely inhibited by NAD. While dehydrogenase activity of the oxidase type is almost negligible, the Vmax value for oxidase activity of this form is similar to that of the dehydrogenase form for dehydrogenase activity, indicating the same rate limiting step of urate release from the enzyme. It should be noted that the dehydrogenase produces more ratio of O2 formation than the oxidase if NAD is absent. However, O2 is almost completely inhibited in the presence of NAD. In contrast to the mammalian enzyme, conversion of the dehydrogenase form to the oxidase form has never been reported for the avian enzyme, but the reactivity of chicken liver xanthine dehydrogenase with oxygen is similar to that of rat liver xanthine dehydrogenase.

Purification of hepatic xanthine dehydrogenase from chicken fed a high-protein diet.

An inhibitor of xanthine oxidase (XO)/xanthine dehydrogenase (XDH), an enzyme catalyzing the last step of purine catabolism, might be expected to be effective as remedy for hyperuricemia and possibly for ischemia-reperfusion injury. However, no clinically effective XO/XDH inhibitor except allopurinol have been used since it was introduced for clinical use in 1962 (1, 2).

Studies on chicken liver xanthine dehydrogenase with reference to the problem of non-equivalence of FAD moieties

Xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.2.1.37 of high specific activity was obtained from chicken liver. As a starting material the livers of chicken fed a high-protein diet were adopted. n nIn the absorption spectrum of the purified enzyme the ratio A280nm/A450nm was 5.54, which suggests a high purity. The specific activity of the purified enzyme was 1200 and 57 as expressed by the turnover number and the activity protein ratio respectively.

Purine nucleoside phosphorylase from bovine liver

1. Reduction of chicken liver xanthine dehydrogenase (xanthine: NAD+ oxidoreductase, EC 1.2.1.37) by xanthine under anaerobic condition proceeded in two phases. This biphasicity may be due to functional and non-functional enzymes in the enzyme preparation. 2. Cyanolysis of a persulfide group of chicken liver enzyme resulted in an inactivation of the enzyme. The non-functional enzyme in the standard enzyme preparation was found to lack persulfide groups at the active sites. 3. The remaining NADH-Methylene Blue oxidoreductase activity, after KI treatment of the xanthine-reduced enzyme of a high flavin activity ratio, is not at the level of 50% of the initial activity, differing from the report suggesting non-equivalence of FAD chromophores. 4. The findings in the present report indicate that FAD chromophores of chicken liver enzyme are essentially equivalent.

Effect of dietary protein on purine nucleoside phosphorylase and xanthine dehydrogenase activities of liver and kidney in chicken and pigeon

1. Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, E.C. 2.4.2.1) from liver of cattle, Bos taurus, was purified to homogeneity. Some properties of the enzymes from three different bovine tissues were compared and discussed. 2. The enzyme has a molecular weight of 83,000, a sedimentation coefficient of 5.3 S, a Stokes radius of 3.71 nm, a frictional ratio of 1.30 and a subunit molecular weight of 30,000. 3. Optimal pH for xanthosine degradation is around 5.5, whereas a broad pH activity profile for inosine degradation was observed between 5.0 and 7.5. Lineweaver-Burk plots curved downward at high concentrations of substrates, inosine, phosphate and arsenate.

Reversible Interconversion Between Sulfo and Desulfo Xanthine Dehydrogenase

Comparative studies were made on the effects of diets of different protein contents on the activities of purine nucleoside phosphorylase and xanthine dehydrogenase of avian livers and kidneys. In chicken liver and kidney, both enzyme activities were increased with high protein diet, confirming the previous results. In pigeon liver, only purine nucleoside phosphorylase was increased but xanthine dehydrogenase activity was not detected after feeding a high protein diet, while both enzyme activities were increased in the pigeon kidney. The increase in the levels of plasma oxypurines in pigeon serum was consistent with the result that the xanthine dehydrogenase activity of pigeon was not detected in the liver but in the kidney.