Rat diaphorase

Example 41

Two fold serial dilutions of PC3 cells were made in F12K media with 10% FBS. 25 ul of each dilution was transferred into wells of a 384-well plate. Detection reagent was made by adding 10 U/ml rat diaphorase (Sigma) and 40 μM proluciferin substrate PBI 4312 into Luciferin Detection Reagent (LDR; Promega Cat. No V8920). 25 μl of detection reagent was added directly to the cells. The reactions were incubated for 30 minutes at room temperature, and luminescence measured using a Tecan plate luminometer.

FIG. 32 shows the correlation between cell number and luminescence indicating a direct relationship between luminescence generated via proluciferin conversion and the total amount of reduced dinucleotides NADH/NADPH present in the cells. The results also show that detection system comprised of diaphorase and pro-luciferin PBI 4312 combined with Luciferase Detection Reagent (LDR) can be added directly to the cells to measure the amount of reduced dinucleotides present in the cells.

Example 33

Each substrate (PBI-4312, 4550 and 4565) was incubated at 20 μM with 10 μM NADH (Sigma) and 5 u/ml rat diaphorase (Sigma) in a volume of 10 μl in 50 mM Tris pH 7.5. Forty identical reactions were prepared. At the timepoints indicated in Table 5, 10 μl LDR with 500 μM menadione was added to 4 of the reactions for each compound. Luminescence was measured at as previously described. RLUs of quadruplicate reactions were averaged (Table 5 and FIG. 22a). Each average signal was also divided by the maximum signal obtained with that compound to calculate the fraction of maximum signal (FIG. 22b).

Example 38

Two fold serial dilutions of NAD (Sigma) were made in PBS starting from 0.25 μM. 25 μl of each dilution was transferred into wells of a 384-well plates. Detection reagent was made by adding 10 U/ml rat diaphorase (Sigma), 40 μM proluciferin substrate PBI 4312 and NAD dependent enzyme amplification system consisting of 5 U/ml Lactate Dehydrogenase (Calbiochem) and 40 mM lactate (Sigma) into Luciferin Detection Reagent (LDR; Promega Cat. No V8920). 25 μl of the detection reagent was added to the NAD samples. The reactions were incubated for 30 minutes at room temperature, and luminescence measured using a Tecan plate luminometer.

The following example demonstrates the use of the pro-luciferin substrate PBI-4312 to detect and measure NAD Luminescence generated is indicative of the presence of NAD with the light output directly proportional to the amount of NAD present in the sample. The results show that the diaphorase and lactate dehydrogenase enzymes remain active in the detection reagent, and all three enzymatic reactions can occur simultaneously (FIG. 30A)

Example 32

Each substrate (PBI-4312, 4550 and 4565) was incubated at 20 μM with 10 μM NADH (Sigma) and 5 u/ml rat diaphorase (Sigma) in a volume of 10 μl 50 mM Tris pH 7.5. Eight identical reactions for each substrate were prepared. The samples were incubated at 5 minutes at room temperature, and 4 of the reactions received 10 μl LDR, and the other 4 reactions received 10 μl LDR with 500 μM menadione. Luciferin generation was monitored over the course of 5 minutes to monitor menadione's inhibition of the diaphorase reaction with each of the 3 substrates. Luminescence was measured, and the RLUs of quadruplicate reactions averaged (Table 4 and FIG. 21). The first reading was taken at approximately one minute after addition of LDR, and the last reading was taken 5 minutes later (˜6 minutes).