Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose.
The oxidase form of xanthine dehydrogenase (XO; EC 1.1.3.22) has been purified approximately 200-fold from rat liver extracts using a three-step process of heat treatment, ammonium sulfate precipitation, and chromatography on benzamidine-Sepharose. The purified enzyme showed only minor contamination when analyzed by gel electrophoresis under either native or sodium dodecyl sulfate (SDS)-denatured conditions and appears to be intact based on its subunit size on SDS-polyacrylamide gel electrophoresis, its N-terminal amino acid sequence, and its ability to be converted to the NAD-dependent dehydrogenase form (XD; EC 1.1.1.204) by incubation with dithiothreitol. Isoelectric focusing analysis showed that the purified enzyme consists of two major, enzymatically active isoforms with average pI values of 6.13 and 6.23 and a minor enzymatically active isoform with an average pl value of 6.07. A similar purification of XD was achieved by preincubating the partially purified oxidase with dithiothreitol prior to affinity chromatography on benzamidine-Sepharose. The effects of benzamidine on the kinetic properties of purified rat XO were characterized at pH 8 and 9 and were compared to those of bovine milk XO. Benzamidine was found to be a weak competitive inhibitor of the purified rat enzyme with Ki values of 30 and 10 mM at pH 8 and 9, respectively. In contrast, the Ki values for benzamidine with bovine XO were more than 10-fold greater. The findings presented in this study show that benzamidine is a competitive inhibitor of XO and that affinity chromatography on benzamidine-Sepharose provides a simple, rapid, and effective means of purifying both the oxidase and dehydrogenase forms of rat XO.
McManaman
J L
JL
Department of Biochemistry, Biophysics, University of Colorado Health Sciences Center, Denver 80262, USA.
Shellman
V
V
Wright
R M
RM
Repine
J E
JE
D042931:Aldehyde Oxidase; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001550:Benzamidines; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004791:Enzyme Inhibitors; D066298:In Vitro Techniques; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D012685:Sepharose; D014968:Xanthine Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014969:Xanthine Oxidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001550:Benzamidines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D012685:Sepharose; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D000445:Aldehyde Oxidoreductases; D042931:Aldehyde Oxidase; C032157:benzamidine