After a membrane suspension (10 mg of protein per mL) is mixed with 20 mmol L-1 EDTA for 30 min in an ice bath. The excess EDTA is removed from the membrane by ultracentrifugation two or three times at 68 000 x g for 60 min. The precipitate is resuspended in a buffer to wash EDTA out of the membrane fraction, followed by ultracentrifugation again under the same conditions. The resulting precipitate is resuspended with the same buffer. Under these conditions, as mentioned below, many PQQ-dependent dehydrogenases are resolved to apoen-zymes. However, some PQQ-dependent dehydrogenases are still active, although some decrease in enzyme activity is observed. If no loss of enzyme activity is found, the presence of covalently bound FAD as the coenzyme is the alternative possi-bility. To convert the apoenzyme to the holoenzyme, PQQ and divalent cations such as Ca2+ or Mg2+ are added to 5 |imol L-1 and 5 mmol L-1, respectively, and the enzyme incubated, for example, for 30 min at 25 °C, until full enzyme activity returns.
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