Sulfhydryl oxidase-catalyzed conversion of xanthine dehydrogenase to xanthine oxidase

Xanthine oxidase may be isolated from various mammalian tissues as one of two interconvertible forms, viz., a dehydrogenase (NAD⁺ dependent, form D) or an oxidase (O₂ utilizing, form O). A crude preparation of rat liver xanthine dehydrogenase (form D) was treated with an immobilized preparation of crude bovine sulfhydryl oxidase. Comparison of the rates of conversion of xanthine dehydrogenase to the O form in the presence and absence of the immobilized enzyme indicated that sulfhydryl oxidase catalyzes such conversion. These results were substantiated in a more definitive study in which purified bovine milk xanthine oxidase, which had been converted to the D form by treatment with dithiothreitol, was incubated with purified bovine milk sulfhydryl oxidase. Comparison of measured rates of conversion (in the presence and absence of active sulfhydryl oxidase and in the presence of thermally denatured sulfhydryl oxidase) revealed that sulfhydryl oxidase enzymatically catalyzes the conversion of type D activity to type O activity in xanthine oxidase with the concomitant disappearance of its sulfhydryl groups. It is possible that the presence or absence of sulfhydryl oxidase in a given tissue may be an important factor in determining the form of xanthine-oxidizing activity found in that tissue.     

A new purification procedure for bovine milk xanthine oxidase: effect of proteolysis on the subunit structure.

A new purification procedure for bovine milk xanthine oxidase is reported. The enzyme so obtained is of the highest purity and shows little evidence of degradation. The enzyme displays a single protein band on either polyacrylamide gels or on sodium dodecyl sulfate-urea polyacrylamide gels. Sedimentation equilibrium studies indicate a native molecular weight of 303,000 and a subunit molecular weight of approximately 150,000. The latter value is in good agreement with the minimum molecular weight of 157,000 calculated from dry weight determination and flavin analysis. In contrast, purification of xanthine oxidase from pancreatin-treated cream yields a protein which displays two subunits corresponding to molecular weights of 92,000 and 39,000 as determined by dodecyl sulfate-urea polyacrylamide gel electrophoresis. Pancreatinized enzyme has a greater mobility than unproteolyzed enzyme on polyacrylamide gels. Exposure of milk xanthine oxidase to pancreatin before isolation or after purification yields the same result.     

A molybdopterin-free form of xanthine oxidase

A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.     

Mammalian xanthine oxidoreductase - mechanism of transition from xanthine dehydrogenase to xanthine oxidase

Reactive oxygen species are generated by various biological systems, including NADPH oxidases, xanthine oxidoreductase, and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide. Recent X-ray crystallographic and site-directed mutagenesis studies have revealed a highly sophisticated mechanism of conversion from XDH to XO, suggesting that the conversion is not a simple artefact, but rather has a function in mammalian organisms. Furthermore, this transition seems to involve a thermodynamic equilibrium between XDH and XO; disulfide bond formation or proteolysis can then lock the enzyme in the XO form. In this review, we focus on recent advances in our understanding of the mechanism of conversion from XDH to XO.     

A superfusion bioassay for platelet-activating factor

A superfusion bioassay for platelet-activating factor is described using various types of tissues. By washing the tissue with 0.1-0.5% bovine serum albumin for 2-3 min after each addition of platelet-activating factor, desensitization did not develop in most tissues studied. Because of the ability to apply a sample directly onto an assay tissue with negligible dilution, this bioassay can detect smaller amounts of platelet-activating factor than those previously reported in which an organ bath was utilized. The ascending colon of the rat and dog appeared to be the most sensitive of the tissues tested, with a limited of detectability in the range of 100-500 fg. Repeated additions of platelet-activating factor could be made for up to 4 h without desensitization. Release of platelet-activating factor from samples of rat stomach was measured using the superfusion bioassay and a platelet aggregation bioassay. There was a significant correlation (r = 0.96; p less than 0.01) between the values obtained using the two assay systems. Thus, the sensitivity, the reproducibility, and the inexpensive nature of this bioassay make it an attractive alternative to existing bioassays for platelet-activating factor.     

Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase

Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR) and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD's redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 Angstroms resolution showed the enzyme's two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favours the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.     

A histochemical method for detecting xanthine oxidase activity in the liver

A histochemical method is described for localizing xanthine oxidase--the key enzyme in the purine catabolic pathway. The above method is based on the reduction of p-Nitroblue tetrazolium during hypoxanthine enzymatic oxidation, phenazine methosulfate being an intermediate electron acceptor. The patterns of the reaction product distribution suggest that the Kupffer cells and the sinusoidal endothelium possess the highest xanthine oxidase activity.     

Multiple molecular forms of xanthine dehydrogenase and related enzymes. IV. The relationship of aldehyde oxidase to xanthine dehydrogenase

Variants of the enzyme aldehyde oxidase in Drosophila melanogaster are described. In addition to electrophoretic variants, a mutant that causes low levels of the enzyme has been found by screening more than 80 strains for aldehyde oxidase levels. The locus of the mutation maps on the third chromosome near lpo and aldox. The existence of the ry, lpo, and aldox mutants and of the new mutant indicates that xanthine dehydrogenase, pyridoxal oxidase, and aldehyde oxidase are under a separate genetic control, in addition to a common genetic control by ma-l and lxd. The genetic separation is shown to be accompanied by physical separation of the enzymes with DEAE-cellulose column chromatography and (NH ₄)₂SO₄fractionation. Further data on the metabolism of aldehydes by xanthine dehydrogenase and aldehyde oxidase are presented. Although xanthine dehydrogenase requires NAD or a similar cofactor to metabolize purine and pteridine substrates, aldehyde oxidase oxidizes salicylaldehyde to salicylic acid without dissociable cofactors and with the uptake of oxygen.This work was supported in part by Research Grant GM-08202, by a Predoctoral Fellowship (J.C.) and a Genetics Training Grant (J.C. and E.D.), and by a Research Career Development Award (E.G.), all from the National Institutes of Health. Part of this work was submitted by J.C. to the University of North Carolina at Chapel Hill in partial fulfillment of the degree of Doctor of Philosophy.