TABLE 1.
Concentrations of various additions to lysis buffer for optimal C. neoformans (1,3)β-glucan synthase activitya
| Addition | Concn tested | Optimal concn |
|---|---|---|
| MgCl2 (mM) | 1-10 | 5 |
| CaCl2 (mM) | 1-10 | 7 |
| NaF (mM) | 1-5 | 2 |
| Phosphatase inhibitor (% [vol/vol]) | 0.01-1 | 0.1 |
| Glycerol (% [vol/vol]) | 10-50 | 20 |
| Sucrose (M) | 0.1-1.0 | 0.5 |
| PMSF (mM) | 0.05-1 | 0.1 |
| DTT (mM) | 1-100 | 1 |
| Protease inhibitor (% [vol/vol]) | 0.01-1 | 0.1 |
| BSA (mg/ml) | 1-5 | |
| EDTA (mM) | 1-100 | 50 |
| EGTA (mM) | 1-100 | 20 |
| GTPγS (μM) | 5-200 | 60 |
a
H99 cells were lysed as described in Materials and Methods, and the optimal concentration of each addition to the lysis buffer resulting in maximal activity was determined as described in Materials and Methods and the text.