Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

Kinetic mechanism of chicken liver xanthine dehydrogenase.

Human inter-alpha-trypsin inhibitor (I alpha I) is a plasma proteinase inhibitor active against cathepsin G, leucocyte elastase, trypsin and chymotrypsin. It owes its broad inhibitory specificity to tandem Kunitz-type inhibitory domains within an N-terminal region. Sequence studies suggest that the reactive-centre residues critical for inhibition are methionine and arginine. Reaction of I alpha I with the arginine-modifying reagent butane-2,3-dione afforded partial loss of inhibitory activity against both cathepsin G and elastase but complete loss of activity against trypsin and chymotrypsin. Reaction of I alpha I with the methionine-modifying reagent cis-dichlorodiammineplatinum(II) resulted in partial loss of activity against cathepsin G and elastase but did not affect inhibition of either trypsin or chymotrypsin. Employment of both reagents eliminated inhibition of cathepsin G and elastase. These findings suggest that both cathepsin G and elastase are inhibited at either of the reactive centres of I alpha I. Trypsin and chymotrypsin, however, appear to be inhibited exclusively at the arginine reactive centre.     

Kinetic isotope effect studies on milk xanthine oxidase and on chicken liver xanthine dehydrogenase

The effect of isotopic substitution of the 8-H of xanthine (with 2H and 3H) on the rate of oxidation by bovine xanthine oxidase and by chicken xanthine dehydrogenase has been measured. V/K isotope effects were determined from competition experiments. No difference in H/T(V/K) values was observed between xanthine oxidase (3.59 +/- 0.1) and xanthine dehydrogenase (3.60 +/- 0.09). Xanthine dehydrogenase exhibited a larger T/D(V/K) value (0.616 +/- 0.028) than that observed for xanthine oxidase (0.551 +/- 0.016). Observed H/T(V/K) values for either enzyme are less than those H/T(V/K) values calculated with D/T(V/K) data. These discrepancies are suggested to arise from the presence of a rate-limiting step(s) prior to the irreversible C-H bond cleavage step in the mechanistic pathways of both enzymes. These kinetic complexities preclude examination of whether tunneling contributes to the reaction coordinate for the H-transfer step in each enzyme. No observable exchange of tritium with solvent is observed during the anaerobic incubation of [8-3H]xanthine with either enzyme, which suggests the reverse commitment to catalysis (Cr) is essentially zero. With the assumption of adherence to reduced mass relationships, the intrinsic deuterium isotope effect (Dk) for xanthine oxidation is calculated to be 7.4 +/- 0.7 for xanthine oxidase and 4.2 +/- 0.2 for xanthine dehydrogenase. By use of these values and steady-state kinetic data, the minimal rate for the hydrogen-transfer step is calculated to be approximately 75-fold faster than kcat for xanthine oxidase and approximately 10-fold faster than kcat for xanthine dehydrogenase. This calculated rate is consistent with data obtained by rapid-quench experiments with XO. A stoichiometry of 1.0 +/- 0.3 mol of uric acid/mol of functional enzyme is formed within the mixing time of the instrument (5-10 ms). The kinetic isotope effect data also permitted the calculation of the Kd values [Klinman, J. P., & Mathews, R. G. (1985) J. Am. Chem. Soc. 107, 1058-1060] for substrate dissociation, including all reversible steps prior to C-H bond cleavage. Values calculated for each enzyme (Kd = 120 microM) were found to be identical within experimental uncertainty.     

NADH oxidase activity of rat liver xanthine dehydrogenase and xanthine oxidase-contribution for damage mechanisms

The involvement of xanthine oxidase (XO) in some reactive oxygen species (ROS) -mediated diseases has been proposed as a result of the generation of O*- and H2O2 during hypoxanthine and xanthine oxidation. In this study, it was shown that purified rat liver XO and xanthine dehydrogenase (XD) catalyse the NADH oxidation, generating O*- and inducing the peroxidation of liposomes, in a NADH and enzyme concentration-dependent manner. Comparatively to equimolar concentrations of xanthine, a higher peroxidation extent is observed in the presence of NADH. In addition, the peroxidation extent induced by XD is higher than that observed with XO. The in vivo-predominant dehydrogenase is, therefore, intrinsically efficient at generating ROS, without requiring the conversion to XO. Our results suggest that, in those pathological conditions where an increase on NADH concentration occurs, the NADH oxidation catalysed by XD may constitute an important pathway for ROS-mediated tissue injuries.     

Nonlinear kinetics of the xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase

The xanthine oxidase reaction catalyzed by chicken liver xanthine dehydrogenase has been shown to give nonlinear kinetics of the type which has been identified as substrate activation. When a very wide range of substrate (pteridine) concentrations were studied, it was found that a downward deflection in reciprocal plots (substrate activation) occurs in the high region and an upward deflection in the very low region. When product (isoxanthopterin) was included in reaction mixtures, the upward deflection was enhanced and shifted to higher substrate concentration ranges. In addition, reciprocal plots with a second substrate (oxygen) and a product (isoxanthopterin) were nonlinear.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Functional groups and quaternary structure of chicken liver xanthine dehydrogenase

Xanthine dehydrogenase from chicken liver is a dimeric enzyme, each hemimolecule containing one FAD and two Fe/S groups. Determination of sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuribenzoic acid (PMB) showed a variable number of sulfhydryl groups depending onpH, ionic strength, and nature of the reaction medium and buffer. The number of disulfide bonds was determined with DTNB and reducing conditions. Amino groups were determined with 2,4,6,-trinitrobencensulfonic acid (TNBS). At constant temperature andpH the reaction of DTNB and TNBS with native xanthine dehydrogenase showed an exponential dependence on time. From the obtained parameters the number of available sulfhydryl and amino groups at infinite concentration of enzyme and the rate constant of the equation were determined. The absorption spectrum of the enzyme changed with time when a chaotropic agent (1 M sodium nitrate) was added to the medium. This difference was detected by measuring the absorbance in the range 450–550 nm. The absorption spectrum (between 350 and 600 nm) also changed when a denaturating agent (sodium dodecyl sulfate) was added. This modification increased with time and depended on the medium.     

Reactivity of chicken liver xanthine dehydrogenase containing modified flavins

Native FAD was removed from chicken liver xanthine dehydrogenase (XDH) and replaced with a number of artificial flavins of different redox potential. Dithionite titration of the 2-thio-FAD- or 4-thio-FAD (high potential)-containing enzymes showed that the first center to be reduced was the flavin. With native enzyme, iron-sulfur centers are the first to be reduced. With the low potential flavin, 6-OH-FAD, the enzyme-bound flavin was the last center to be reduced in reductive titration with xanthine. These shifts in the reduction profile support the hypothesis that the distribution of reducing equivalents in multi-center oxidation-reduction enzymes of this type is determined by the relative potentials of the centers. The reaction of molecular oxygen with fully reduced 2-thio-FAD XDH or 4-thio-FAD XDH resulted in 5 electron eq being released in a fast phase and one in a slow phase. Reduction of these enzymes by xanthine was limited at a rate comparable to that for the release of urate from native XDH. Xanthine/O2 turnover with these enzymes (and native XDH) resulted in approximately 40-50% of the xanthine reducing equivalents appearing as superoxide. Steady state turnover experiments involving all modified flavin-containing enzymes, as well as native enzyme, showed that shifting the flavin potential either positive or negative relative to FAD caused a decrease in catalytic activity in the xanthine/NAD reductase reaction. In the case of the xanthine/O2 reductase activity, there is no simple obvious relationship between the activity and the redox potential of the reconstituted flavin.     

N-ethylmaleimide inhibits xanthine oxidase activity with no detectable change in xanthine dehydrogenase activity in rabbit liver

The effect of an alkylating agent, N-ethylmaleimide (NEM), on the activities of xanthine oxidase (XO) and xanthine dehydrogenase (XD) in the presence and absence of Cu2+ or trypsin in the cytosolic fraction from rabbit liver was examined. At concentrations ranging from 0.25 to 2.0 microM, allopurinol, which is generally considered to be a XO inhibitor, suppressed the XD activity (41.5-93.4% inhibition) in addition to the XO activity (28.6-88.4% inhibition) under basal conditions, without the addition of Cu2+ or trypsin. In contrast, NEM (100-400 microM) inhibited the XO activity (35.7-85.7% inhibition) without affecting the XD activity. Also, NEM inhibited the Cu2+- and trypsin-induced XO activities, but did not affect the XD activity at the same concentration range. These results demonstrate that NEM can be a selective inhibitor of XO activity in rabbit liver.