The NAD⁺ kinase activity assay is a pivotal biochemical technique used to measure the activity of NAD⁺ kinase, an enzyme responsible for phosphorylating NAD⁺ to produce NADP⁺. This conversion is crucial for maintaining the balance of the NAD/NADP⁺ pool, which plays a significant role in various metabolic processes, including cellular respiration, lipid synthesis, and the regulation of oxidative stress.

The assay typically involves incubating a reaction mixture containing NAD⁺, ATP, and the enzyme source under controlled conditions. Following incubation, the reaction is terminated, and the resulting NADP⁺ can be quantified using spectrophotometric methods or high-performance liquid chromatography (HPLC). One common approach to this assay is to couple the reaction with glucose-6-phosphate dehydrogenase (G6PDH), where the production of NADP⁺ leads to the reduction of NAD⁺ to NADH. 

This assay is particularly valuable in research focused on metabolic regulation, cancer biology, and studying the effects of various compounds on cellular metabolism. Additionally, understanding NAD⁺ kinase activity has implications for therapeutic strategies targeting metabolic disorders and age-related diseases, making it an essential tool in both basic and applied research.