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Preparative Nondenaturing Gel Electrophoresis to Purify NADP-Specific Glutamate Dehydrogenase From Chlorella, Rev B

by operating the unit in a 4C coldroom. The protein sample, 68 mg total protein in 9 ml of loading buffer, was loaded on top of the stacking gel and electrophoresed for 20 min at 15 mA, and then for an additional 3.5 hr at a constant power of 30 mA using Bio-Rads Model 3000/300 power supply.

Fraction Collection and Analysis
The elution buffer was pumped at a rate of 2 ml/min to a fraction collector and 6 ml fractions were collected. The first fraction after the Bromophenol Blue marker eluted, fraction 1, was collected after 2 hr of electrophoresis. A spectrophotometric assay was used to quantitate the deaminating activity of the NADP-GDH in each fraction. One unit of GDH activity was defined as the amount of enzyme activity required to reduce 1 mol of NADP+/min at 38.5C. NADP-GDH activity was detected in fractions 3568 (Figure 1). The multiple peaks of enzyme activity detected presumably correspond to the multiple isoenzymes of the NADP-GDH.

Analysis of Model 491 Prep Cell Purified NADP-GDH
NADP-GDH containing fractions were combined and the protein concentration was determined by the method of Bradford (1976) using the Bio-Rad protein assay kit with Bio-Rads protein standard II as the standard. Fractions under the peaks were pooled, concentrated, and rinsed with 10 mM KPO4 (pH 6.2), then resuspended in 10 mM KPO4 (pH 6.2), 0.1 mM NADP+ to a concentration of 1 mg/ml for further analysis and storage at 20C. The purity of the protein was determined by Tris-Tricine SDS-PAGE (Schagger and von Jagow 1987) in a 10% polyacrylamide resolving, 3% stacking gel using the Mini-PROTEAN II slab cell (Figure 2). Gels were stained using the Bio-Rad Silv
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