2.8 VIN activity assay
VIN was assayed following the method of Schaffer et al. (1989), with
modifications. Frozen samples of mesocarp (1 g) were homogenized on ice
in 5 mL of extraction buffer containing 100 mM sodium phosphate buffer
(pH 7.5), 5 mM MgCl2, 2.5 mM dithiothreitol, 0.1%
Tritonx-100 (v/v), and 2% polyvinypolypyrrolidone (PVPP, m/v). The
homogenates were centrifuged at 12,000 × g for 20 min at 4 °C.
Supernatants were dialyzed in 0.1 × extraction buffer (without PVPP) to
remove soluble sugar. VIN activity was assayed using the protocol
described above. One unit of VIN activity was defined as the amount of
enzyme required to produce 1 µmol glucose per hour, expressed as U/g
fresh weight.