Identification of the products synthesized by
coleoptiles (a) and seeds (b) using the sarcoplasmic reticulum
Ca2+-ATPase and the PPase from yeast. The assay medium
composition was 50 mm Mops-Tris buffer, pH 7.0, 100
mm KCl, 0.1 mm ADP, 5 mm
MgCl2, 5 mm 32Pi, and 0.05 mg/mL
tonoplast protein. The reactions were started by adding either 1
mm ATP or 1 mm PPi. After 30 min the reactions
were stopped by filtration to remove vesicles, and the filtrates were
divided into four aliquots. Three of them were incubated with 0.05
mg/mL Ca2+-ATPase from skeletal muscle sarcoplasmic
reticulum (hatched bars), 0.01 mg/mL PPase from yeast (gray bars), or
both (white bars); one aliquot (control; black bars) was not incubated
with either enzyme. The Ca2+-ATPase reaction medium was
supplemented with 0.15 mm CaCl2, 2
μm A23187, and 0.1 mm ATP when ATP was not
already present. The PPase reaction medium was supplemented with 0.1
mm PPi when PPi was not already present. The reaction was
stopped after 30 min by the addition of TCA (10%, w/v). The medium was
subjected to phosphomolybdate extraction, and the radioactivity
remaining in the aqueous phase was counted. Values represent the means
+ se of six independent experiments.