Comparison of Mg2+-dependent ATP hydrolase activities of pea nodule symbiosomes and of pea root plasmalemma, obtained by an aqueous polymer two-phase system.
The characteristics of the Mg2+-dependent ATPase activity from the peribacteroid membrane of pea symbiosomes was compared with that from pea root plasma membranes. Enzyme inhibitors, optimum reaction pH, substrate specificity and antibody recognition were the main parameters examined. Both the symbiosomes and the root plasma membrane were purified with an aqueous polymer two-phase system (APS). The final concentration of the APS for the purification of symbiosomes were: 6.3% w/w dextran T500, 6.3% w/w poly(ethylene glycol) 3350, 5 mM KH2PO4-K2HPO4, 5 mM KCl, 0.33 M sucrose, (pH 7.85); for the root plasma membrane was: 6.2% (w/w) dextran T500, 6.2% poly(ethylene glycol) 3350, 330 mM sucrose, 5 mM K2HPO4 and 4 mM KCl (pH 7.8). The lack of contamination of pea symbiosomes with endoplasmic reticulum, mitochondria, broken bacteroids and/or tonoplast vesicles was established. Similarly, the aqueous two-phase system used here provided a fairly enriched root plasma membrane with low cross-contamination from other sources. Both symbiosomal and root plasma membrane ATPase activities were highly specific to ATP. The symbiosome ATPase apparently corresponds to an E1E2-ATPase mechanism, similar to that found at the plasma membrane. The similarity between these two ATPases was further supported by immuno-analysis. Mg2+-ATPase of pea symbiosome and root plasma membranes were very similar, by all parameters tested.
Departamento de Química Agrícola, Geología y Geoquímica, Universidad Autónoma de Madrid, Spain.
P
P
Rojas-Ojeda
L E
LE
Hernández
N J
NJ
Brewin
R
R
Carpena-Ruiz
D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism; D015388:Organelles / Q000201:enzymology*; D018532:Pisum sativum / Q000201:enzymology*; D018517:Plant Roots / Q000201:enzymology*; D013559:Symbiosis
D004791:Enzyme Inhibitors; D017301:Ca(2+) Mg(2+)-ATPase; D008274:Magnesium