| Abstract: | NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23) from yeast has been purified utilizing ion-exchange and NAD+-agarose affinity chromatography to give a 2100-fold purification. The apparent homogeneity of the enzyme preparation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a subunit molecular weight of 31,000, and a native molecular weight of 124,000, and is, thus, probably a tetramer. The single form of the enzyme has an apparent isoelectric point of 5.85. Initial velocity studies in the forward direction with both substrates gave intersecting Lineweaver-Burk plots, and this suggests a sequential mechanism in which both substrates are bound before products are released. Replots of these data were linear and gave Km values for NAD+ and ATP of 0.68 mM and 2.3 mM, respectively. |
