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After 30 minutes at 37C, 500 L of cold ethanol was added to the homogenate to precipitate proteins, followed by centrifugation (21,000 g , 4C, 10 minutes), and the supernatant was dried under an N 2 stream at room temperature

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Contribution of some other residues, namely, His401, Thr415, Thr417, E419, and Arg571, reported to be important for proper positioning of Gln398 and Thr399, was also assessed by the same group (Figure 3)