Table 1.
Kinetic characterization of protein fractions with erythrose reduction activity isolated from rabbit liver
Reaction buffer consisted of 50 mm sodium phosphate buffer, pH 6.8, 200 μm NADPH or NADH, and at least 5 concentrations of erythrose ranging from 0.5 to 100 mm. Reactions were initiated with addition of purified protein. Final concentrations of protein were 49 μm fraction A (ADH1) for NADPH, 25 μm fraction A (ADH1) for NADH, 0.7 μm fraction B (SORD) for NADPH, and 2.6 μm fraction B (SORD) for NADH. Kinetic constants were determined for the NAD-dependent conversion of ethanol to acetaldehyde for fraction A (ADH1) by monitoring production of NADH at 340 nm. Kinetic constants were also determined for the NAD-dependent conversion of sorbitol to fructose for fraction B (SORD) by monitoring production of NADH at 340 nm. Reactions were initiated with addition of purified protein (final concentration 1.3 μm for fraction A (ADH) and 0.13 μm for fraction B (SORD)). Measurements at each substrate concentration were performed in duplicate. Data are shown as average and standard deviation determined from fitting the data to Y = Vmax × X/(Km + X), where Y represents reaction velocity and X indicates substrate concentration using GraphPad Prism software. Km values are presented as “apparent Km” because these values were determined at only one saturating concentration of cofactor.
| Substrate | Cofactor | Apparent Km | kcat | kcat/Km | |
|---|---|---|---|---|---|
| mm | s−1 | m−1 s−1 | |||
| Fraction A, ADH1 | Erythrose | NADPH | 64.5 ± 7.7 | 0.0041 ± 0.0003 | 0.064 ± 0.009 |
| Erythrose | NADH | 80.1 ± 18.9 | 0.016 ± 0.002 | 0.205 ± 0.059 | |
| Ethanol (control) | NAD | 5.7 ± 0.5 | 0.106 ± 0.004 | 18.6 ± 1.8 | |
| Fraction B, SORD | Erythrose | NADPH | 340.3 ± 29 | 1.65 ± 0.11 | 4.84 ± 0.53 |
| Erythrose | NADH | 266 ± 120 | 0.13 ± 0.05 | 0.51 ± 0.29 | |
| Sorbitol (control) | NAD | 1.15 ± 0.16 | 0.26 ± 0.01 | 230 ± 33 |