Purification of Nicotinate Phosphoribosyltransferase by Affinity Chromatography

概要

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Nicotinate phosphoribosyltransferase-specific affinity chromatography was prepared from succinylated aminoalkyl agarose coupled with 6-amino nicotinic acid, a competitive inhibitor of nicotinate phosphoribosyltransferase, and was applied to the selective purification of the enzyme of baker's yeast.<BR>Three millimoles of 6-amino nicotinic acid prepared from 6-amino nicotinamide with acid hydrolysis were added to 50ml of succinyldiaminodipropyl agarose at pH 7.4, followed by the addition of three millimoles of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and the reaction was allowed to proceed at 4° for 24 hours. The substituted agarose was then washed continuously with distilled water. About 3 μmoles of 6-amino nicotinic acid were covalently bound per ml of agarose. The enzyme solution was added to the affinity column equilibrated with 10 mM Tris-phosphate buffer (pH 8.0) and the enzyme was then eluted with 0.1 M Tris-phosphate buffer containing 0.1 M ammonium sulfate (pH 8.6). About 75-fold purification was attained by this single step procedure. The capacity to adsorb the enzyme to the substituted agarose was strongly affected by the concentration of proteins other than the enzyme.
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