TABLE 1.
Glucose metabolism by resting cells
| Item | Data seta:
|
||||
|---|---|---|---|---|---|
| 1 | 2 | 3 | 3A | 4 | |
| Strain | DFY724 (wild type) | DFY725 (gcr2) | DFY724 (wild type) | DFY724 (wild type) | DFY725 (gcr2) |
| Substrate employed for growth | Glucose | Glucose | Lactic acid | Lactic acid + glucose (0.75 h) | Lactic acid |
| V of enzyme (μmol/[min × mg of protein])b | |||||
| Zwf | 0.22 ± 0.034 (4) | 0.44 ± 0.17 (3) | 0.33 ± 0.06 (5) | 0.31 | 0.34 ± 0.04 (4) |
| Pgi | 12.5 ± 4.92 (5) | 9.58 ± 3.42 (3) | 8.50 ± 2.18 (4) | 8.95 | 1.68 ± 0.15 (3) |
| Tpi | 41.1 ± 19.2 (3) | 21.1 ± 9.76 (2) | 19.4 | 10.4 | —e |
| Gap | 9.90 ± 2.96 (4) | 8.85 ± 4.08 (3) | 7.82 ± 3.30 (5) | 8.68 | 3.84 ± 1.52 (4) |
| Pgk | 95.9 ± 39.6 (4) | 29.6 ± 8.10 (3) | 54.7 ± 14.9 (5) | 62.9 | 5.16 ± 0.98 (4) |
| Gpm | 10.8 ± 3.84 (5) | 2.35 ± 0.28 (6) | 4.11 ± 2.00 (5) | 5.55 | 0.51 ± 0.11 (4) |
| Eno | 7.93 ± 3.20 (4) | 2.42 ± 0.92 (3) | 2.10 ± 0.56 (5) | 2.22 | 0.16 ± 0.07 (4) |
| Pyk | 10.8 ± 4.81 (4) | 5.40 ± 2.30 (3) | 3.01 ± 0.70 (5) | 4.11 | 0.93 ± 0.21 (4) |
| vglucose (μmol/[min × mg of protein])d | 0.30 ± 0.03 (9) | 0.30 ± 0.03 (10) | 0.09 ± 0.02 (5) | 0.18 | 0.02 ± 0.01 (5) |
| Products (μmol/μmol of glucose used) | |||||
| Ethanol | 1.40 ± 0.36 (8) | 1.28 ± 0.20 (8) | 1.28 ± 0.16 (5) | 1.41 | 0.94 ± 0.37 (4) |
| Glycerol | 0.14 ± 0.05 (7) | 0.25 ± 0.05 (7) | 0.20 ± 0.06 (5) | 0.27 | 0.72 ± 0.30 (4) |
| Intracellular metabolite concn (mM)e | |||||
| Glucose-6-phosphate | 0.06 ± 0.06 (3), 6.36 ± 1.36 (5) | 0.12 ± 0.14 (4), 5.78 ± 1.75 (6) | 1.23 ± 0.24 (3) | 2.20 | 0.79 ± 0.28 (2) |
| Fructose-1,6-bisphosphate | 0.08 ± 0.04 (5), 15.2 ± 3.26 (6) | 0.10 ± 0.10 (5), 12.2 ± 1.97 (6) | 2.88 ± 0.86 (3) | 6.08 | 1.10 ± 0.61 (2) |
| Glycerate-3-phosphate | 0.94 ± 0.24 (5), 0.97 ± 0.19 (6) | 0.92 ± 0.65 (4), 4.31 ± 1.13 (7) | 0.39 ± 0.01 (2) | 1.06 | 7.52 ± 2.71 (2) |
| Glycerate-2-phosphate | 0.18 ± 0.06 (5), 0.15 ± 0.05 (5) | 0.22 ± 0.09 (5), 1.36 ± 0.49 (5) | 0.05 ± 0.02 (3) | 0.08 | 0.64 ± 0.29 (2) |
| Phosphoenolpyruvate | 0.34 ± 0.09 (5), 0.31 ± 0.08 (5) | 0.30 ± 0.14 (5), 0.52 ± 0.22 (5) | 0.04 ± 0.01 (3) | 0.06 | 0.05 ± 0.04 (2) |
| Data seta:
| |||||
|---|---|---|---|---|---|
| 4A | 5 | 5A | 6 | 7 | 8 |
| DFY725 (gcr2) | DFY726 (gcr1) | DFY726 (gcr1) | DFY727 (gpm1) | DFY728 (wild type) | DFY730 (gpm1 pGAL-GPM) |
| Lactic acid + glucose (3 h) | Lactic acid | Lactic acid + glucose (6 h) | Lactic acid | Galactose | Galactose |
| 0.27 | 0.37 ± 0.01 (2) | 0.39 | 0.33 | — | — |
| 4.82 | 0.7 ± 0.18 (2) | 2.58 | 6.52 | — | — |
| 16.3 | 0.54 | 5.38 | — | — | — |
| 7.59 | 0.92 ± 0.28 (2) | 11.96 | 4.17 | — | — |
| 14.1 | 7.92 ± 1.6 (2) | 30.7 | 58.12 | — | — |
| 1.99 | 0.09 ± 0.15 (3) | 0.41 | 0.003 ± 0.004 (2) | 10.1 ± 7.59 (3) | 0.30 ± 0.42 (3) |
| 1.74 | 0.16 ± 0.02 (3) | 0.82 | 2.21 ± 0.16 (2) | 3.86 | 5.03 |
| 4.51 | 0.51 ± 0.01 (2) | 2.08 | 3.77 | — | — |
| 0.16 | 0.03 | 0.10 | 0.0 | 0.25 ± 0.07 (3) | 0.03 ± 0.02 (4) |
| 1.35 | 0.76 | 0.87 | 0.0 | 1.19 | 0.72 |
| 0.33 | 0.92 | 0.33 | 0.0 | 0.12 | 0.64 |
| 3.84 | 0.92 | 2.80 | 0.19 | 7.32 ± 0.96 (2) | 0.78 ± 0.33 (2) |
| 9.72 | 0.56 | 5.60 | 0.08 | 7.24 | 5.72 |
| 6.96 | 8.08 | 13.52 | 17.0 | 0.87 ± 0.24 (2) | 8.36 ± 0.84 (2) |
| 0.80 | 0.15 | 0.04 | — | 0.048 ± 0.02 (2) | 0.85 ± 0.20 (2) |
| 0.16 | 0.01 | 0.06 | — | 0.17 ± 0.18 (2) | 0.27 ± 0.13 (2) |
Throughout the table, values are means ± standard deviations, based on the number of separate experiments indicated in parentheses.
Enzyme abbreviations are as follows: Zwf, glucose-6-phosphate dehydrogenase; Pgi, phosphoglucose isomerase; Tpi, triose phosphate isomerase; Gap, glyceraldehyde-3-phosphate dehydrogenase; Pgk, phosphoglycerate kinase; Gpm, phosphoglycerate mutase; Eno, enolase; and Pyk, pyruvate kinase. Protein was determined by the biuret assay using bovine albumin (Sigma Protein Standard). With the substrate concentrations employed, most of the V values are near to Vmax. Because most of the assays employ the reverse direction to glycolysis, those values were corrected by the indicated Vf/Vr factors (Vf and Vr are the Vmax values for the forward and reverse reactions, respectively): Pgi 2.5 (3, 32); Tpi 3.2 (22); Gap, 1.5 (not yeast [14]); Pgk, 8.9 (5); and Gpm, 0.6 (12).
—, not determined.
Expressed in the same units as enzyme activity, using a value of 79 ± 4.3 μg of protein per ml of cell suspension with an A580 value of 1.0. (The value was obtained with eight strains or conditions corresponding to data sets 1 to 7 in a calibration experiment using trichloroacetic acid precipitation of cells followed by 30 min at 100°C in 1 N NaOH and biuret assay [16]. The same experiment gave a dry weight [15 h at 102°C] of 197 ± 33 μg and a wet weight of 868 ± 212 μg, all per unit A580.)
Intracellular concentrations are given as 40 times that measured in the final neutralized acid extracts (1.382 ml/100 A580 units of cells, as described above), using an internal volume value of 2 μl per mg (dry wt); values of 2 to 2.5 have been reported (1, 13, 18, 32). For data sets 1 and 2, the first and second entries separated by a comma correspond to sample times of −2 min (before the addition of glucose; vglucose = 0.0 μmol/[min × mg of protein]) and 10 min, respectively. The remainder of the data sets had one sampling point each, as follows: 3, 20 min; 3A, 22 min; 4, 20 min; 4A, 22 min; 5, 22 min; 5A, 24 min; 6, 30 min; 7, 10 min; 8, 40 min.