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- W2184269255 abstract "Plasmamembrane preparations ofhighpurity (about 95%)are easily obtained bypartitioning inaqueouspolymer two-phase systems. Thesepreparations, however, mainly contain sealed right-side-out (apoplastic sideout) vesicles. Part ofthese vesicles havebeenturned inside-out byfreezing andthawing, andsealed inside-out andright-side-out vesicles subsequently separated by repeating thephasepartition step. Increasing theKCIconcentration inthefreeze/thaw mediumaswell asincreasing thenumber offreeze/thaw cycles significantly increased theyield ofinsideoutvesicles. Atoptimal conditions, 15to25%oftotal plasma membraneprotein wasrecovered asinside-out vesicles, corresponding to5to10milligrams ofprotein from500gramsofsugar beet(Beta vulgaris L.)leaves. Basedonenzymelatency, trypsin inhibition ofNADH-cytochrome c reductase, andH+pumping capacity, across-contamination ofabout20%between thetwo fractions ofoppositely oriented vesicles wasestimated. Thus, preparations containing about80%inside-out and80% rightside-out vesicles, respectively, wereobtained. ATPaseactivity andH+pumping werebothcompletely inhibited byvanadate (K, ; 10micromolar), indicating thatthefractions werecompletely free fromnonplasma membrane ATPases. Furthermore, thepolypeptide patterns ofthetwofractions wereclosetoidentical, whichshowsthatthevesicles differed insidedness only. Thus, preparations ofbothinside-out andright-side-out plasma membranevesicles arenowavailable. Thispermits studies ontransport, signal transduction mechanisms, enzymetopology, etc., using plasma membranevesicles ofeither orientation." @default.
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- W2184269255 date "1990-01-01" @default.
- W2184269255 modified "2023-09-27" @default.
- W2184269255 title "Optimal Conditions forFormation andSeparation" @default.
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