Purification and properties of deoxyriboaldolase from human erythrocytes

Abstract

Deoxyriboaldolase (2-deoxy-d-ribose-5-phosphate acetaldehyde lyase, EC 4.1.2.4) from human erythrocytes was purified approx. 2500 times by treatment of hemolysates with (NH4)2SO4 (25%) and two successive treatments with calcium phosphate gel. The enzyme was unstable in hemolysates (half life, 2 days) and partially purified preparations, but could be stabilized by 0.01 M MgCl2 (half life, 4 days) or sulfhydrylreagents (dithiothreitol). The latter could also reactivate storage inactivated preparations. Erythrocyte deoxyriboaldolase was activated by dicarboxylic and tricarboxylic acids of which citrate was most effective and activated 2-3 fold. Cis-configuration activators (maleate) were more effective than trans-isomers (fumarate), indicating a proximity requirement for adjacent carboxyl groups of the activator. At several citrate concentrations uncompetitive activation kinetics were observed. Km and vmax for the enzyme without citrate was 96 μM and 0.96 mM/h, and with 4 mM citrate was 361 μM and 4.17 mM/h, respectively. Additionally, citrate caused aggregation of the enzyme. © 1970.