Selective Delipidation of the Plasma Membrane by Surfactants

Abstract

The influence of plasma membrane lipid components on the activity of the H+-ATPase has been studied by determining the effect of surfactants on membrane lipids and ATPase activity of oat (Avena sativa L.) root plasma membrane vesicles purified by a two-phase partitioning procedure. Triton X-100, at 25 to 1 (weight/weight) Triton to plasma membrane protein, an amount that causes maximal activation of the ATPase in the ATPase assay, extracted 59% of the membrane protein but did not solubilize the bulk of the ATPase. The Triton-insoluble proteins had associated with them, on a micromole per milligram protein basis, only 14% as much phospholipid, but 38% of the glycolipids and sterols, as compared with the native membranes. The Triton insoluble ATPase could still be activated by Triton X-100. When solubilized by lysolecithin, there were still sterols associated with the ATPase fraction. Free sterols were found associated with the ATPase in the same relative proportions, whether treated with surfactants or not. We suggest that surfactants activate the ATPase by alterng the hydrophobic environment around the enzyme. We propose that sterols, through their interaction with the ATPase, may be essential for ATPase activity.The factors that influence the activity of the PM2 H+-ATPase have attracted considerable attention because of the importance of this enzyme in solute uptake, membrane potential and plant growth mechanisms (35). The ATPase exists in situ in a lipid environment consisting of phospholipids, sterols, and glycolipids (8,9,21,25,31). It has been assumed that a specific phospholipid environment is required for optimal activity. For example, treatment of membranes with surfactants, which should cause partial delipidation, generally reduces ATPase activity, while readdition of phospholipid mixtures partially restores activity (8,11,19,34).

Comparison of the Lipid Composition of Oat Root and Coleoptile Plasma Membranes

“…Lynch and Steponkus (19) analyzed the lipids from cold-acclimated and nonacclimated rye leaves. Rochester et al (21,22) (2,14,25). Reports exist which state that cyclopropyl sterols may influence the plant PM ATPase activity (2,14).…”

“…Removal of most of the phospholipids from oat root PM ATPase, leaving the enzyme enriched with glycolipid and sterol, resulted in enhanced activity as long as Triton X-100 was present to provide the necessary lipid environment (25). When this fraction was then treated with lysolecithin, the ATPase was solubilized and further enriched in sterols.…”

“…Individual sterols in the neutral lipid samples were quantified by GLC with known plant sterols as standards as previously described (25). Total sterol amounts, calculated by addition of individual sterol amounts determined by gas chromatography, always gave values greater than those determined by the colorimetric method.…”

“…Individual glycolipids were separated by HPTLC, as described previously (25), identified by coelution with known standards after spraying with TNS (16), scraped from the plate and analyzed by the above procedure. Two solvent systems were used to develop glycolipid samples on HPTLC plates (Merc) to help identify the most polar (unknown) glycolipids found in this fraction.…”

Exogenous DNA Uptake via Cellular Permeabilization and Expression of Foreign Gene in Wheat Zygotic Embryos.

“…The embryos treated with common membrane destabiliser DMSO (20%) failed to exhibit histochemical GUS expression and also lowered the percentage of embryo germination (Table 1), in contrast to that reported by Topfer et al (1989). Similarly, Triton X-100 (0.1%), a popular membrane surfactant involved in selective delipidation of membranes and also used to extract Fig membrane proteins (Sandstorm and Cleland, 1989), was found inefficient for DNA uptake. Triton X-100 also had a detrimental effect on embryo germination greater than any other membrane permeabilizer.…”