The extract was filtered
through two layers of Miracloth and centrifuged at 30,000 g for
15 min at 4 C. A 2.5 mL sample of the supernatant was then loaded
into a Sephadex G-25 column (PD 10; Pharmacia, Madrid, Spain)
that had previously been equilibrated with 10 mL phosphate buffer
(pH 6). The enzyme was eluted with 3.5 mL of the same buffer.