The
homogenate was filtered through two layers of Miracloth and
centrifuged at 20,000 g for 15 min at 4 C and then a 2.5 mL
aliquot was loaded into a Sephadex G-25 column (PD 10; Pharmacia,
Madrid, Spain) equilibrated with 10 mL 0.1 M phosphate
buffer (pH 7.8). The enzymes were eluted with 3.5 mL of the same
buffer. The resulting supernatant was used as an enzyme extract to
determine enzyme activity.