Abstract
Abstract Nicotinamide adenine dinucleotide kinase (ATP:NAD+2'-phosphotransferase, EC 2.7.1.23) has been purified 20-fold from extracts of unfertilized sea urchin eggs. The pH optimum for activity was 6.5. The enzyme was specific for ATP, and a divalent cation was required for activity. At 1:1 cation/ATP ratios, relative activities were Mn2+ g Mg2+ g Ca2+. Free Mn2+ was inhibitory, whereas activation by free Ca2+ was observed. Complex kinetic behavior was observed when the concentration of one substrate was varied in the presence of a high level of the second substrate. Activation was observed with increasing concentrations of NAD+ and ATP. Substrate concentrations giving one-half maximum velocities were 0.83 mm ATP and 0.68 mm NAD+. No activation was seen with a variety of metabolites. NADPH, NADH, ADP, AMP, and cyclic 3',5'-AMP were inhibitors. The molecular weight estimated by gel filtration and density gradient centrifugation was 310,000. The relationship between the properties of the enzyme and the changes in pH, cation content, and substrate levels in eggs at fertilization were considered.
Published Version
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