On the presence of inside‐out plasma membrane vesicles and vanadate‐inhibited K+,Mg2+‐ATPase in microsomal fractions from wheat and maize roots

Abstract

We have estimated the amount of inside‐out plasma membrane (PM) vesicles in microsomal fractions from wheat (Triticum aestivum L. cv. Drabant) and maize (Zea mays L.) roots; non‐latent activities of the PM markers vanadate‐inhibited K+, Mg2+‐ATPase (ΔVO4‐ATPase) and glucan synthase II (GS II, EC 2.4.1.34) were used as markers for inside‐out PM vesicles, latent activities as markers for right‐side‐out PM vesicles, and specific staining with silicotungstic acid (STA) as a general marker for the PM. Separation of presumptive inside‐out PM vesicles from right‐side‐out ones was achieved by counter‐current‐distribution (CCD) in an aqueous polymer two‐phase system. Most of the GS II activity was latent and was found in material partitioning into the upper phase; a distribution which correlated well with that of STA‐stained vesicles. Thus, most of the PM vesicles had a right‐side‐out orientation. ΔVO4‐ATPase, on the other hand, had a dual distribution (particularly pronounced in wheat) and was recovered both in material partitioning into the lower phase and into the upper phase. This indicates that ΔVO4‐ATPase activity was present also in membranes other than the PM. Additional evidence for this interpretation came from sucrose gradient centrifugation of wheat root material. This produced two peaks of ΔVO4‐ATPase activity with the membranes partitioning into the lower phase, none of which coincided with the peak obtained with right‐side‐out PM vesicles. Taken together, these results indicate that only very few inside‐out PM vesicles are present in the microsomal fraction, and that ΔVO4‐ATPase as a marker for the PM, in contrast to GS II, may give quite misleading results with some plant materials. This stresses the need to use well‐defined preparations of scaled, inside‐out PM vesicles in solute uptake studies. The distribution of Ca2+‐inhibited ATPase, on the other hand, agreed well with those of GS II and STA‐stained vesicles both after CCD and sucrose gradient centrifugation, which suggests that Ca2+ inhibition may be a more specific property of the PM H+‐ATPase than vanadate inhibition.