Isolation and characterization of yeast nicotinamide adenine dinucleotide kinase.
Biochimica et biophysica acta
confidence
Key findings
Purified yeast NAD+ kinase to 2100-fold; subunit MW 31,000, native 124,000 (tetramer); isoelectric point 5.85; sequential mechanism; Kms 0.68 mM NAD+, 2.3 mM ATP.
View source on PubMed (PMID 221029) ↗
- Population
- Yeast (in vitro enzyme purification)
- Blinding
- not_reported
- Controls
- none
- Drug class
- coenzyme
Full 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.