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Purification and properties of milk xanthine dehydrogenase.
J Biol Chem. 1992 Oct 25; 267(30):21479-85.JB

Abstract

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.

Authors+Show Affiliations

Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

1328233

Citation

Hunt, J, and V Massey. "Purification and Properties of Milk Xanthine Dehydrogenase." The Journal of Biological Chemistry, vol. 267, no. 30, 1992, pp. 21479-85.
Hunt J, Massey V. Purification and properties of milk xanthine dehydrogenase. J Biol Chem. 1992;267(30):21479-85.
Hunt, J., & Massey, V. (1992). Purification and properties of milk xanthine dehydrogenase. The Journal of Biological Chemistry, 267(30), 21479-85.
Hunt J, Massey V. Purification and Properties of Milk Xanthine Dehydrogenase. J Biol Chem. 1992 Oct 25;267(30):21479-85. PubMed PMID: 1328233.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification and properties of milk xanthine dehydrogenase. AU - Hunt,J, AU - Massey,V, PY - 1992/10/25/pubmed PY - 1992/10/25/medline PY - 1992/10/25/entrez SP - 21479 EP - 85 JF - The Journal of biological chemistry JO - J Biol Chem VL - 267 IS - 30 N2 - Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/1328233/Purification_and_properties_of_milk_xanthine_dehydrogenase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)36634-7 DB - PRIME DP - Unbound Medicine ER -