Table 1.
Relative activities of GAPDH in the presence of tTGase, various Q donors and GAPDH substrates
| Minus GAPDH substrate
|
Plus GAPDH substrate
|
||
|---|---|---|---|
| 1.5 mM GAP | 1.0 mM NAD+ | ||
| Experiment 1 | |||
| Q donor added | |||
| Control (minus tTGase) | [100 ± 4] | – | – |
| None | 83 ± 3 | – | – |
| CGG (25 mM) | 35 ± 5 | 65 ± 2* | 101 ± 2* |
| Casein (4 mg/ml) | 48 ± 1 | 80 ± 5* | 90 ± 6* |
| GSTQ0 (1.6 mg/ml) | 43 ± 1 | 45 ± 1 | 72 ± 2* |
| GSTQ10 (1.2 mg/ml) | 53 ± 1 | 60 ± 2* | 73 ± 2* |
| GSTQ62 (1.7 mg/ml) | 6 ± 1 | 35 ± 1* | 78 ± 2* |
| Experiment 2 | |||
| Q donor added | 8.2 ± 2 | 16 ± 3* (0.3 mM GAP) | |
| GSTQ62 (1.7 mg/ml) | 21 ± 10 (0.03 mM GAP) | ||
| 19 ± 4* (0.1 mM NAD+) | |||
| 22 ± 4* (0.01 mM NAD+) | |||
| 64 ± 2* (1.0 mM NADH) | |||
| 19 ± 4* (0.1 mM NADH) | |||
| 22 ± 4* (0.01 mM NADH) | |||
The reaction mixture contained 100 mM Tris⋅HCl buffer (pH 7.6), 10 mM CaCl2, 10 mM DTT, GAPDH (0.8 μg), tTGase (10 milliunits/units; specific activity of 2 units/mg of protein) and the indicated additions in a final volume of 20 μl. After 1 h at 20°C, 2-μl aliquots were withdrawn and assayed for GAPDH activity in a well plate analyzer. n = three or four separate determinations. All values in the “minus GAPDH substrate” column are significantly different from the control (i.e., minus tTGase) with a P = 0.05.
*
Significant protection against the corresponding value obtained in the absence of added GAPDH substrate with P = 0.05.