Table 2.
Composition of reagent mixtures for all enzyme assaysa
| Enzyme | Reaction mixture components |
|---|---|
| GLKb (EC 2.7.1.2) | 4 mM MgSO4, 2 mM ATP, 0.4 mM NADP+, 5 U · ml−1 G6PDH and initiator: 10 mM glucose |
| G6PDHb (EC 1.1.1.49) | 2 mM MgSO4, 0.4 mM NADP+ and initiator: 10 mM glucose-6P |
| PGI (EC 5.3.1.9; physiological direction) | 7 mM MgSO4, 5 mM ATP, 0.3 mM NADH, 1 U · ml−1 PFK, 1 U · ml−1 ALD, 2 U · ml−1 G3PD, 5 U · ml−1 TPI, and initiator: 10 mM glucose-6P |
| PGI (nonphysiological direction) | 2 mM MgSO4, 0.4 mM NADP+, 1.75 U · ml−1 G6PDH and initiator: 20 mM fructose-6P |
| PFK (EC 2.7.1.11) | 7 mM MgSO4, 5 mM ATP, 0.3 mM NADH, 1 U · ml−1 ALD, 2 U · ml−1 G3PD, 5 U · ml−1 TPI, and initiator: 20 mM fructose-6P |
| ALD (EC 4.1.2.13) | 2 mM MgSO4, 0.3 mM NADH, 2 U · ml−1 G3PD, 5 U · ml−1 TPI, and initiator: 30 mM fructose-1,6BP |
| TPI (EC 5.1.3.1) | 2 mM MgSO4, 0.3 mM NADH, 2 U · ml−1 G3PD, and initiator: 6 mM glyceraldehyde-3P |
| GAPDH (EC 1.2.1.12) | 5 mM MgSO4, 5 mM cysteine-HCl, 50 mM potassium phosphate, 3 mM ADP, 14.5 U · ml−1 PGK, 5 mM NAD+ and initiator: 10 mM glyceraldehyde-3P |
| PGK (EC 2.7.2.3; nonphysiological direction) | 7 mM MgSO4, 5 mM ATP, 0.3 mM NADH, 8 U · ml−1 GAPDH, and initiator: 10 mM 3-PGA |
| PGM (EC 5.4.2.1) | 5 mM MgSO4, 3 mM ADP, 0.1 mM 2,3BPG, 0.3 mM NADH, 2 U · ml−1 ENO, 5 U · ml−1 PYK, 10 U · ml−1 LDH, and initiator: 5 mM 3P-glycerate |
| ENO (EC 4.2.1.11; PEP absorbancec) | 5 mM MgSO4, 3 mM ADP, 0.3 mM NADH, 5 U · ml−1 PYK, 10 U · ml−1 LDH, and initiator: 5 mM 2P-glycerate; 2 mM MgSO4, and initiator: 5 mM 2P-glycerate |
| PYK (EC 2.7.1.40) | 5 mM MgSO4, 3 mM ADP, 5 mM fructose-1,6BP, 0.3 mM NADH, 10 U · ml−1 LDH, and initiator: 6 mM PEP |
| LDH (EC 1.1.1.27) | 2 mM MgSO4, 3 mM fructose-1,6BP, 0.3 mM NADH, and initiator: 6 mM PYR |
| ACK (EC 2.7.2.1) | 5 mM MgSO4, 3 mM ADP, 2 mM glucose, 0.4 mM NADP+, 8.5 U · ml−1 hexokinase, 12.7 U · ml−1 G6PDH, and initiator: 5 mM acetyl-P |
| PTA (EC 2.3.1.8; PTA control) | 2 mM MgSO4, 0.08 mM DTNB, and initiator: 0.4 mM acetyl-coenzyme A. 2 mM MgSO4, 0.008 mM DTNB, 2 mM acetyl-P, and initiator: 0.4 mM acetyl-CoA |
| ADH (EC 1.1.1.1) | 2 mM MgSO4, 0.3 NADH, and initiator: 20 mM acetaldehyde |
| ALDHd (EC 1.2.1.10; nonphysiological direction) | 2 mM MgSO4, 0.1 mM CoA, 1 mM DTT, 0.5 mM NAD+, and initiator: 40 mM acetaldehyde |