dehydratase was unchanged by the detergent treatment, but this step was necessary to

remove lipid vesicles. The 12,000 x g centrifugation that followed the detergent

treatment resulted in a decrease in the specific activity of diol dehydratase.

Table 4-1. Diol dehydratase activity during organelle purification
                Protein       Activity      Specific                   Fold 
Sample          (mg)          (U)a          activity      Yield ()     purification
                                            (U/mg)
Crude extract   500.0         1,346          2.7          100.0         1.0
Detergent/salts 370.6         1,000          2.7           74.3         1.0
treatment
12,000 x g      357.0           573          1.6           42.6         0.6
super
48,000 x g        9.4           194         20.8           14.4         7.7
pellet
12,000 x g        7.0           195         27.9           14.5        10.3
super
Sucrose
density           0.6            16         27.5            1.2        10.2
gradient
aOne unit activity is defined as the amount of enzyme that catalyzes the formation of 1 [tmol of propionaldehyde per min per mg protein.

      However, this step was needed to remove aggregates, which included many

organelles that were not released from vesicles by the detergent treatment. Following

aggregate removal, the polyhedral organelles were pelleted by centrifugation at 48,000 x

g, resuspended in buffer, and clarified by a second centrifugation at 12,000 x g. These

two steps proved very effective and increased the specific activity of diol dehydratase

from 1.6 to 27.9 [tmol/min/mg. The final sucrose density gradient step typically resulted

in a slight drop in the specific activity of diol dehydratase (Table 4-1). The reason is

uncertain. It may have been due to removal of an unknown factor that enhances diol

dehydratase activity or due to partial enzyme inactivation.

      For the complete purification, the specific activity of diol dehydratase increased

approximately 10-fold, from 2.7 to 27.5 [tmol/min/mg protein (Table 4-1). This indicates