Glutamate Dehydrogenase Isolation
Chlorella sorokiniana cells were cultured in 29 mM NH4+ medium as previously described (Baker and Schmidt 1963). Approximately 130 g fresh weight cells were harvested by centrifugation and washed two times in 0.01 M Tris-HCl (pH 8.5, 4C). Pelleted cells were resuspended in an equal ratio of breakage buffer (w/v) and ruptured by two passages through a French pressure cell (American Inst. Co.) at 20,000 psi. The cell homogenate was centrifuged at 27,000 x g for 45 min and the supernatant was stored overnight at 20C. Frozen supernatant was thawed and the resulting precipitate was removed by centrifugation at 27,000 x g. Initial purification of the NADP-GDH was accomplished using a modified procedure of Yeung et al. (1981), which employs sequential ion exchange and size exclusion chromatography to remove the bulk contaminating proteins from the cell lysate. Procedural modifications involved the addition of NADP+, which functioned as a stabilizer, to a final concentration of 0.1 mM to the gel filtration buffer and all subsequent buffers. As a final modification, a preparative nondenaturing PAGE step using the Model 491 prep cell was substituted for an expensive NADP-affinity resin step.
Following ammonium sulfate precipitation and ion exchange chromatography, size exclusion column fractions, in 10 mM KPO4, 2 mM dithiothreitol (DTT), 0.1 mM NADP+ (pH 6.2), possessing NADP-GDH activity were pooled and concentrated via Diaflo filtration from 17.5 ml to 5 ml. The soluble enzyme was reduced by the addition of DTT to a final concentration of 10 mM and pla