Table I.
Purification Step | Total Protein | Specific Activity | Total Activity | Purification | Recovery |
---|---|---|---|---|---|
mg | pkat g−1 | pkat | -fold | % | |
(NH4)2SO4 | 56 | 0.071 | 0.004 | - | 100 |
Superose-12 | 20 | 0.81 | 0.016 | 11.5 | 35.7 |
Mono-Q | 4 | 3.26 | 0.013 | 46 | 7.2 |
Adenosine-agarose | 0.12 | 13.8 | 0.0017 | 194 | 0.21 |
Leaves (approximately 20 g) were extracted with Pi buffer, the homogenate filtered and centrifuged, and the protein that precipitated between 30% and 70% ammonium sulfate saturation was desalted on PD-10 column before successive chromatography on Superose-12, Mono-Q, and adenosine-agarose columns.
Enzyme assays were carried out using Q as the substrate, as described in the “Materials and Methods” section.