Table 2.

Binding of [³⁵S]PSK-α to various centrifugation fractions of rice cell membranes

Centrifugation		Specific binding, fmol/mg protein	Marker enzyme activity, nmol/ min per mg protein
Force	Time, min		Vanadate-sensitive ATPase	Cyt-c oxidase	NADPH–Cyt-c reductase
1,000  × g	10	8.4	9.4	2.7	2.0
8,000  × g	15	27.2	13.9	15.7	2.4
13,000  × g	15	44.7	28.9	23.0	2.1
40,000  × g	30	60.1	33.3	22.0	2.9

Rice cell homogenate was filtered and successively centrifuged at various forces. The membrane pellets were suspended in binding buffer and aliquots (400 μl) of membrane suspensions were incubated with 32 nM [³⁵S]PSK-α for 240 min at 4°C in the absence or presence of 100-fold excess of unlabeled PSK-α. After incubation, the reaction mixture was overlaid onto 1.0 ml of binding buffer containing 0.5 M sucrose and centrifuged at 86,000 × g for 5.0 min at 4°C. The supernatant was discarded and the pellet was analyzed for radioactivities. Marker enzymes used were as follows: vanadate-sensitive ATPase for plasma membrane, cytochrome-c oxidase for mitochondria, and NADPH–cytochrome-c reductase for endoplasmic reticulum.