( by operating the unit in a 4C coldroom...)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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