Plasma membrane vesicles were isolated from shoots of light‐grown wheat seedlings by preparative free‐flow electrophoresis, aqueous polymer two‐phase partition or both. Plasma membrane vesicles were identified from staining of thin sections prepared for electron microscopy with phosphotungstic acid at low pH. The orientation of the plasma membrane vesicles was determined from latency and trypsin sensitivity of K+ Mg2+ATPase and of glucan synthase II, and concanavalin A‐peroxidase binding and membrane asymmetry visualized by electron microscopy. The K+Mg2+ATPase and of glucan synthase II activities of plasma membrane fractions isolated by two‐phase partition were latent and trypsin resistant. The vesicles bound concanavalin A‐peroxidase strongly and exhibited a cytoplasmic side‐in morphology. These fractions of cytoplasmic side‐in vesicles were less than 10% contaminated by cytoplasmic side‐out vesicles. By free‐flow electrophoresis, two populations of vesicles which stained with phosphotungstic acid at low pH, designated D and E, were obtained. The vesicle population with the lower electrophoretic mobility, fraction E, contained plasma membrane vesicles with properties similar to those of the plasma membrane vesicles obtained after two‐phase partition. The phosphotungstic‐reactive vesicles with greater electrophoretic mobility, fraction D, were concanavalin A unreactive with the cytoplasmic membrane leaflet outwards. Less than 50% of the K+Mg2+‐ATPase activity of this fraction was latent and trypsin sensitive. The vesicles of fraction D appeared to be preferentially cytoplasmic side‐out. The electrophoretic mobilities of cytoplasmic side‐out (non‐latent glucan synthase II activity) and cytoplasmic side‐in (latent glncan synthase II activity) plasma membrane vesicles isolated from a frozen and thawed wheat plasma membrane fraction, corresponded with the mobilities of fraction D and E, respectively, again showing that the plasma membrane vesicles with the lesser electrophoretic mobility were cytoplasmic side‐in. The cytoplasmic side‐in and cytoplasmic side‐out vesicles therefore showed opposite eletrophoretic mobilities compared with a previous free‐flow electrophoretic separation of soybean plasma membranes. The majorities of the plasma membrane vesicles of both fractions D and E entered the upper phase upon two‐phase partition with the phase composition used for purification of wheat plasma membranes. Thus, neither electrophoretic mobility nor phase partitioning characteristics can be used as the only criteria for assignment of vesicle orientation.

Comparing isolated plasma membrane vesicles and excised hypocotyl segments from etiolated seedlings of soybean [Glycine max (L.) Merr. cv. Williams], certain antiproliferative agents that inhibited growth inhibited plasma membrane redox activities. Additionally, auxins that stimulated growth stimulated plasma membrane redox activities. Hormone stimulation was restricted to NADH oxidase (determined from disappearance of NADH) and was given both by isolated plasma membranes and by a soluhilizedenzyme preparation. Comparing IAA, the native auxin regulator, and 2,4‐D, a synthetic regulator, stimulation was observed, hut the dose‐response curves were different. Yet, the dose‐response relationships of both stimulation of auxin growth and stimulation of NADH oxidase were parallel. Inhibition of auxin‐induced growth by antiproliferative drugs was more complex. Some, like actinomycin D, preferentially inhibited NADH oxidase (EC 1.6.99.2) but inhibited NADH‐ferricya‐nide oxido‐reductase (EC 1.6.99.3) as well. Others, like adriamycin, inhibited primarily the NADH‐ferricyanide oxido‐reductase. Therefore, growth control by auxin appeared to involve NADH oxidase as a rate‐limiting terminal oxidase to link electron flow from NADH to oxygen. This observation may provide a fundamental difference from animal cells. With the latter, impermeant electron acceptors such as diferric transferrin or ferricyanide fulfill such a role. In plants, these impermeant electron acceptors were without effect on growth or were growth inhibitory.