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Plasma membrane vesicles of opposite sidedness from soybean hypocotyls by preparative free-flow electrophoresis.
MedLine Citation:
PMID:  16665959     Owner:  NLM     Status:  PubMed-not-MEDLINE    
Abstract/OtherAbstract:
Absolute orientations (sidedness) of plasma membrane vesicles obtained in highly purified fractions by preparative free-flow electrophoresis and by aqueous two-phase partition were determined based on ATPase latency and morphological criteria. Free-flow electrophoresis yielded two plasma membrane fractions. One, the least electronegative and designated fraction ;E,' was pure plasma membrane. The other, more electronegative and designated fraction ;C,' was heavily contaminated by various other cellular membranes. Plasma membrane vesicles from both fraction C and fraction E partitioned into the upper phase with aqueous two-phase partitioning. Purified plasma membrane obtained from microsomes by two-phase partition (upper phase) when subjected to free-flow electrophoresis also yielded two fractions, one fraction co-migrated with fraction C and another fraction co-migrated with fraction E. Both fractions exhibited an ATPase activity sensitive to vanadate and insensitive to nitrate and azide. ATPase activity was used as a structure-linked latency marker for the inner membrane surface. Concanavalin A binding (linked to peroxidase) was used as an imposed electron microscope marker for the outer membrane surface. Fraction E vesicles showed low ATPase latency (two-fold or less) and weak reactivity with concanavalin A peroxidase. In contrast, fraction C vesicles were characterized by much greater latencies upon detergent treatment (sevenfold) and a strong reaction with concanavalin A peroxidase. Two-phase partition as the initial procedure for plasma membrane isolation, yielded mixtures of vesicles of both inside out and right-side out orientation. Free-flow electrophoresis resolved the plasma membrane isolates into vesicles from fraction C which were right-side out (cytoplasmic side in), and vesicles from fraction E which were wrong-side out (cytoplasmic side out). Therefore, the two methods used in series, provided highly purified membrane preparations of apparently homogenous vesicles of opposite known absolute orientations.
Authors:
H Canut; A Brightman; A M Boudet; D J Morré
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Plant physiology     Volume:  86     ISSN:  0032-0889     ISO Abbreviation:  Plant Physiol.     Publication Date:  1988 Feb 
Date Detail:
Created Date:  2010-06-29     Completed Date:  2010-06-29     Revised Date:  2010-09-14    
Medline Journal Info:
Nlm Unique ID:  0401224     Medline TA:  Plant Physiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  631-7     Citation Subset:  -    
Affiliation:
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
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