Preparation of corn root plasmalemma with low Mg-ATPase latency and high electrogenic H+ pumping activity after phase partitioning

Crude plasma membranes of corn (Zea mays L.) roots were obtained according to MI De Michelis and RM Spanswick (1986 Plant Physiol 81: 542-547). This preparation, which contained tightly sealed vesicles displaying Mg-ATP dependent H+-transport, was purified by phase partitioning. The percentage of in...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1988-06, Vol.87 (2), p.491-497
Main Authors: Galtier, N, Belver, A, Gibrat, R, Grouzis, J.P, Rigaud, J, Grignon, C
Format: Article
Language:English
Subjects:
Active sites
Adenosine triphosphatases
Biological and medical sciences
CATION
CATIONES
CATIONS
Cell membranes
Cell physiology
CELL STRUCTURE
Charge density
Corn
Dyes
ESTRUCTURA CELULAR
Fundamental and applied biological sciences. Psychology
General aspects, methods
HIDROGENO
HIDROLASAS
HYDROGEN
HYDROGENE
HYDROLASE
HYDROLASES
MAGNESIO
MAGNESIUM
Membrane potential
Membranes and Bioenergetics
Microsomes
NUTRICION DE LAS PLANTAS
NUTRITION DES PLANTES
PLANT NUTRITION
Plant physiology and development
Plant roots
PROTON PUMP
RACINE
RAICES
ROOTS
STRUCTURE CELLULAIRE
Washing
ZEA MAYS
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Summary:Crude plasma membranes of corn (Zea mays L.) roots were obtained according to MI De Michelis and RM Spanswick (1986 Plant Physiol 81: 542-547). This preparation, which contained tightly sealed vesicles displaying Mg-ATP dependent H+-transport, was purified by phase partitioning. The percentage of inside-out vesicles (10%) was determined from the Mg-ATPase latency, revealed with lysophosphatidylcholine. A Triton X-100 treatment described previously (JP Grouzis, R Gibrat, J Rigaud, C Grignon 1987 Biochim Biophys Acta 903: 449-464) was applied to phase-partitioned plasma membranes. The percentage of catalytic sites freely accessible to Mg-ATP increased to 50% after Triton X-100 treatment. Treated vesicles remained capable of electrogenic H+-pumping, as demonstrated by Mg:ATP-dependent quinacrine fluorescence quenching and oxonol absorbance shift. As expected from the large increase of the catalytic sites accessibility, increases of the dye responses were observed. Concanavalin A binding was estimated from microelectrophoretic measurements of individual vesicles. Statistical analysis of concanavalin A binding and Mg-ATPase latency suggest that treated membranes have lost their asymmetric structure.
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.87.2.491