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Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose.
Arch Biochem Biophys. 1996 Aug 01; 332(1):135-41.AB

Abstract

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.

Authors+Show Affiliations

Department of Biochemistry, Biophysics, University of Colorado Health Sciences Center, Denver 80262, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

8806718

Citation

McManaman, J L., et al. "Purification of Rat Liver Xanthine Oxidase and Xanthine Dehydrogenase By Affinity Chromatography On Benzamidine-sepharose." Archives of Biochemistry and Biophysics, vol. 332, no. 1, 1996, pp. 135-41.
McManaman JL, Shellman V, Wright RM, et al. Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose. Arch Biochem Biophys. 1996;332(1):135-41.
McManaman, J. L., Shellman, V., Wright, R. M., & Repine, J. E. (1996). Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose. Archives of Biochemistry and Biophysics, 332(1), 135-41.
McManaman JL, et al. Purification of Rat Liver Xanthine Oxidase and Xanthine Dehydrogenase By Affinity Chromatography On Benzamidine-sepharose. Arch Biochem Biophys. 1996 Aug 1;332(1):135-41. PubMed PMID: 8806718.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification of rat liver xanthine oxidase and xanthine dehydrogenase by affinity chromatography on benzamidine-sepharose. AU - McManaman,J L, AU - Shellman,V, AU - Wright,R M, AU - Repine,J E, PY - 1996/8/1/pubmed PY - 1996/8/1/medline PY - 1996/8/1/entrez SP - 135 EP - 41 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 332 IS - 1 N2 - 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. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/8806718/Purification_of_rat_liver_xanthine_oxidase_and_xanthine_dehydrogenase_by_affinity_chromatography_on_benzamidine_sepharose_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(96)90325-2 DB - PRIME DP - Unbound Medicine ER -