Fig. 10

Inactivation of the trypsin/salt extract for reconstitution of trypsin/salt-treated adipocyte.

Isolated rat adipocytes desensitized for PIG-P action by combined trypsin/salt treatment were supplemented with 200 µl of trypsin/salt extract (t/s), which had been pretreated with heat, proteinase K (PK), or NEM, or dialyzed (dial.) or left untreated (see Materials and Methods). Alternatively, extracts were pretreated with V8 protease from Staphylococcus aureus in the presence of 0.5 M NaCl (v/s) or with 0.5 M NaCl alone (s). After reconstitution, the cells were washed twice by flotation, incubated in the absence or presence of PIG-P (10 µm) as indicated and then assayed for lipogenesis. Each bar represents the mean ± SEM of three different preparations of the extract and reconstitution reactions, with lipogenesis measurements in quadruplicate. The stimulation factor for lipogenesis in adipocytes reconstituted with untreated trypsin/salt extract in the absence of PIG-P was set at 1.