Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts
A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic component...
Gespeichert in:
Veröffentlicht in: | Plant physiology (Bethesda) 1980-07, Vol.66 (1), p.187-193 |
---|---|
Hauptverfasser: | , , |
Format: | Artikel |
Sprache: | eng |
Schlagworte: | |
Online-Zugang: | Volltext |
Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
Zusammenfassung: | A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross-contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1-5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis. |
---|---|
ISSN: | 0032-0889 1532-2548 |
DOI: | 10.1104/pp.66.1.187 |