Quinolinic Acid Phosphoribosyltransferase from Castor Bean Endosperm: I. PURIFICATION AND CHARACTERIZATION

Abstract

Abstract Quinolinic acid phosphoribosyltransferase was purified 500-fold from the endosperm of etiolated seedlings of Ricinus communis L. Sodium dodecyl sulfate disc gel electrophoresis indicated that the enzyme was homogeneous but on analytical disc gel electrophoresis at pH 7.0 and 8.3 the isolated native protein exhibited three closely migrating bands. The three proteins were of the same molecular weight but differed slightly in charge, determined by Ferguson plots of the Rf values of the proteins in gels having different acrylamide concentrations. The purified enzyme was stable for several months when stored at -90° in 0.05 m potassium phosphate buffer (pH 7.0) which contained 50% sucrose (w/v) and was 0.01 m in dithioerythritol. The molecular weight of the enzyme was estimated by gel titration and sucrose density gradient centrifugation to be about 68,000 and 72,000, respectively. Electrophoresis on sodium dodecyl sulfate polyacrylamide gels demonstrated that the enzyme contained two subunits with a molecular weight of approximately 35,000 each. Quinolinic acid at 0.8 mm gave 50% protection against heat inactivation of the enzyme, but phosphoribosylpyrophosphate at 1.6 mm was ineffective. Inhibition of quinolinic acid phosphoribosyltransferase by several substrate analogues suggested that both the 2- and the 3-carboxyl groups on the pyridine ring were needed for substrate binding to the enzyme. The kinetic data of the reaction are consistent with the formation of a ternary complex among the enzyme, quinolinic acid, and 5-phosphoribosyl 1-pyrophosphate.