Most of the plasma membrane vesicles formed upon homogenization of plant tissue have a right-side-out (cytoplasmic side-in) orientation. Subsequent purification of plasma membrane vesicles using aqueous two-phase partitioning leads to a further enrichment in right-side-out vesicles resulting in preparations with 80-90% of the vesicles in this orientation. Thus, to be able to assay, e.g. the ion-pumping activities of the H(+)-ATPase and the Ca(2+)-ATPase, which expose their active sites towards the cytoplasm, the vesicles have to be inverted. This is very efficiently achieved by including 0.05% of the detergent Brij 58 (C16E20) in the assay medium, which produces 100% sealed, inside-out (cytoplasmic side-out) vesicles from preparations of 80-90% right-side-out vesicles. This was shown by assaying ATP-dependent H+ pumping using the delta pH probe acridine orange and dissipating the H+ gradient with nigericin, and by assaying ATP-dependent Ca2+ transport using 45Ca2+ and dissipating the Ca2+ gradient with the ionophore A23187. The presence of intact vesicles was confirmed by electronmicroscopy. The detergent Brij 58 is a polyoxyethylene acyl ether and a survey among some other members of this series revealed that those with a head group of relatively large size (E20-23) showed this 'non-detergent behavior', whereas those with smaller head groups (E8-10) behaved as normal detergents and permeabilized the membranes. Thus, a very convenient system for studies on ion-pumping activities and other vectorial properties of the plasma membrane is obtained by simply including the detergent Brij 58 in the assay medium.

Plasma membrane preparations of high purity were obtained from roots of dark‐grown wheat (Triticum aestivum L. cv. Drabant) by aqueous polymer two‐phase partitioning. These preparations mainly contained sealed, right‐side‐out vesicles (ca 90% exposing the original outside out). By subjecting the preparations to 4 freeze/thaw cycles the proportion of sealed, inside‐out (cytoplasmic side out) vesicles increased to ca 30%. Inside‐out and right‐side‐out plasma membrane vesicles were then separated by partitioning the freeze/thawed plasma membranes in another aqueous polymer two‐phase system. In this way, highly purified, sealed, inside‐out (>60% inside‐out) vesicles were isolated and subsequently used for characterization of the Ca2+ transport system in the wheat plasma membrane. The capacity for 45Ca2+ accumulation, nonlatent ATPase activity and proton pumping (the latter two markers for inside‐out plasma membrane vesicles) were all enriched in the inside‐out vesicle fraction as compared to the right‐side‐out fraction. This confirms that the ATP‐binding site of the 45Ca2+ transport system, similar to the H+‐ATPase, is located on the inner cytoplasmic surface of the plant plasma membrane. The 45Ca2+ uptake was MgATP‐dependent with an apparent Km for ATP of 0.1 mM and a high affinity for Ca2+ [Km(Ca2+/EGTA) = 3 μM]. The pH optimum was at 7.4–7.8. ATP was the preferred nucleotide substrate with ITP and GTP giving activities of 30–40% of the 45Ca2+ uptake seen with ATP. The 45Ca2+ uptake was stimulated by monovalent cations; K− and Na+ being equally efficient. Vanadate inhibited the 45Ca2+ accumulation with half‐maximal inhibitions at 72, 57 and 2 μM for basal, total (with KCI) and net K+‐stimulated uptake, respectively. The system was also highly sensitive to erythrosin B with half‐maximal inhibition at 25 nM and total inhibition at 1μM. Our results demonstrate the presence of a primary Ca2+ transport ATPase in the plasma membrane of wheat roots. The enzyme is likely to be involved in mediating active efflux (ATP‐binding sites on the cytoplasmic side) to the plant cell exterior to maintain resting levels of cytoplasmic free Ca2+ within the cell.

The spinach (Spinacia oleracea L.) leaf plasma membrane Ca2+‐ATPase is regulated by calmodulin (3‐fold stimulation) and limited proteolysis (trypsin; 4‐fold stimulation). The plasma membrane Ca2+‐ATPase was identified as a 120‐kDa polypeptide on western immunoblots using two different antibodies. During trypsin treatment the 120‐kDa band diminished and a new band appeared at 109 kDa. The appearance of the 109‐kDa band correlated with the increase in enzyme activity following trypsin treatment. The stimulations by calmodulin and trypsin were not additive, suggesting that the 109‐kDa polypeptide represents a Ca2+‐ATPase lackin a terminal fragment involved in calmodulin regulation. This was confirmed by 125I‐calmodulin overlay studies where calmodulin labeled the 120‐kDa band in the presence of Ca2+, while the 109‐kDa band did not bind calmodulin. The effects of calmodulin and limited proteolysis on ATP‐dependent accumulation of 45Ca2+ in isolated inside‐out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca2+ and ATP. Calmodulin increased the Vmax. for Ca2+ pumping 3‐fold, and reduced Km for Ca2+ from 1.6 to 0.9 µM. The Km for ATP (11 µM) was not affected by calmodulin. The effects of limited proteolysis on the affinities for Ca2+ and ATP were similar to those obtained with calmodulin. Notably, however, limited proteolysis increased the Vmax. for Ca2+ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.

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The effects of the phytotoxin prehelminthosporol from the fungal pathogen Bipolaris sorokiniana on the activities of the plasma membrane H+‐ATPase, Ca2+‐ ATPase, and 1, 3‐β‐glucan synthase in barley roots were investigated in vitro. Plasma membranes were isolated by aqueous polymer two‐phase partitioning. Proton pumping by the H+‐ATPase was drastically reduced in a dose‐dependent manner, with complete inhibition at 500 μM prehelminthosporol, while inhibition of ATP hydrolysis was less drastic, with a 35% inhibition at 500 μM toxin. Ca2+ uptake was also reduced, although to a lesser extent than proton pumping, with a maximal inhibition of 60% at 500 μM toxin. The 1, 3‐β‐glucan synthase activity was weakly stimulated at toxin concentrations below 100 μM, with higher concentrations being inhibitory. Taken together, our results indicate that prehelminthosporol exerts its effect in at least two ways. First, it appears to disrupt the membrane barrier and thus seriously interfere with establishment of the proton gradient that drives ion and nutrient uptake and affect the efflux of Ca2+ to maintain the low cytoplasmic concentration of Ca2+ essential for the function of Ca2+ as a messenger in signal transduction. Secondly, it appears to inhibit the enzyme activities. The 1, 3‐β‐glucan synthase is activated by Ca2+ and can operate in the presence of low concentrations of prehelminthosporol (which may induce Ca2+ leakage into the cytoplasm and hence activate the enzyme), to produce callose to seal leaky membranes.