Figure 4.

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.