TABLE 4.
Effect of vanadate on the ATPase activity of MalFGK(KK42N) and the isolated MalK subunit
| Protein sample | Conditions
|
Relative ATPase activity ± vanadate in assayab | Relative ATPase activity following preincubation ± vanadatebc | |
|---|---|---|---|---|
| MBP | Vanadate concn (mM) | |||
| MalFGK(KK42N) | + | 0 | 1.0 | 1.0 |
| MalFGK(KK42N) | + | 0.1 | 0.11 | 0.94 |
| MalK | − | 0 | 1.0 | 1.0 |
| MalK | − | 0.1 | 0.85 | NDd |
| MalK | − | 0.5 | 0.75 | 0.94 |
ATPase activity of proteoliposomes containing MalFGK(KK42N) was assayed at 37°C in 20 mM HEPES buffer (pH 8) with 5 μM MBP, 0.1 mM maltose, 10 mM MgCl2 and 1 mM ATP in the presence or absence of vanadate. MalK was assayed at 37°C with 3 mM MgCl2, 1 mM ATP, 20 mM HEPES (pH 8), 50 mM NaCl, 20% glycerol, and 0.1% lauryldimethylamine oxide in the presence or absence of the indicated concentration of vanadate.
ATPase activities of the uninhibited samples are 0.1 μmol/min/mg for MalK and 0.04 μmol/min/mg for MalFGK(KK42N) with MBP present. These values are normalized to 1, and data from the treated samples are presented as a fraction of the uninhibited values. Each point is the mean of three determinations that varied within 0.15 of the mean. Without MBP present, the MalFGK(KK42N) preparation hydrolyzed only 0.002 μmol ATP/min/mg.
Proteoliposomes containing MalFGK(KK42N) were preincubated for 20 min at 37°C in 20 mM HEPES buffer (pH 8) containing 10 mM MgCl2, 5 μM MBP, and 1 mM ATP in the presence or absence of vanadate and then diluted and collected by centrifugation to remove vanadate. MalK was incubated for 20 min at 23°C in buffer containing 20 mM HEPES (pH 8), 3 mM MgCl2, 1 mM ATP, 50 mM NaCl, 20% glycerol, and 0.1% lauryldimethylamine oxide in the presence or absence of the indicated concentration of vanadate and then subjected to gel filtration prior to assay.
ND, not determined.