The isolation of demolybdo xanthine oxidase from bovine milk.
It was deduced many years ago from indirect evidence that demolybdo xanthine oxidase is present in normal bovine milk. This has now been confirmed by isolation of this enzyme form by a method based on the folate-gel affinity-chromatography procedure described Nishino & Tsushima [(1986) J. Biol. Chem. 261, 11242-11246]. Enzymic and spectroscopic properties of demolybdo xanthine oxidase, which retains flavin and iron-sulphur centres, are generally in accordance with expectations. Like the normal enzyme, it yields on denaturation material fluorescing at 460 nm. Molybdenum cofactor activity measured by the Neurospora crassa nit-1 assay in the presence of added molybdate was 33% of that of the normal enzyme. The absorption spectrum in the near-u.v. region differs slightly, but significantly, from that of the active and desulpho forms of the enzyme. It is concluded that the molybdenum cofactor site contains a pterin-like material not identical with that in the normal enzyme. The significance of the occurrence of demolybdo xanthine oxidase in milk is discussed, and evidence in the literature for demolybdo forms of other molybdoenzymes is briefly reviewed. Additional studies on the use of the affinity procedure for large-scale preparation of high-activity xanthine oxidase are described. In agreement with our ability to isolate the demolybdo enzyme, the procedure appears less effective in eliminating the demolybdo than the desulpho enzyme.
Ventom
A M
AM
School of Chemistry and Molecular Sciences, University of Sussex, Falmer, Brighton, U.K.
Deistung
J
J
Bray
R C
RC
D000818:Animals; D002846:Chromatography, Affinity; D003067:Coenzymes / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D008667:Metalloproteins / Q000378:metabolism; D008892:Milk / Q000201:enzymology*; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D009243:NAD / Q000494:pharmacology; D011621:Pteridines / Q000378:metabolism; D013053:Spectrophotometry; D014969:Xanthine Oxidase / Q000302:isolation & purification* / Q000378:metabolism
D003067:Coenzymes; D005182:Flavin-Adenine Dinucleotide; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D009243:NAD; D011621:Pteridines; D014969:Xanthine Oxidase; C022261:molybdopterin