The properties of the plasma membrane H+-ATPase and the cause of its latency have been studied using a highly purified plasma membrane fraction from oat (Avena sativa L, cv Victory) roots, prepared by aqueous two-phase partitioning. The ATPase has a maximum specific activity (at 37°C) in excess of 4 micromoles inorganic phosphate per milligram protein per minute in the presence of nondenaturing surfactants. It is inhibited by more than 90% by vanadate, is specific for ATP, has a pH optimum of 6.5, and is stimulated more than 4-fold by 50 millimolar K' in the presence of low levels of the nondenaturing surfactants Triton X-100 and lysolecithin. This 'latent' activity is usually explained as being a result of the inability of ATP to reach the ATPase in right-side out, sealed vesicles, until they are disrupted by surfactants. Consistent with this idea, trypsin digestion significantly inhibited the ATPase only in the presence of the surfactants. Electron spin resonance spectroscopy volume measurements confirmed that surfactant-free vesicles were mostly sealed to molecules similar to ATP. However, the Triton to protein ratio required to disrupt vesicle integrity completely is 10-fold less than that needed to promote maximum ATPase activity. We propose that plasma membrane ATPase activation is due not solely to vesicle disruption and accessibility of ATP to the ATPase but to the surfactants activating the ATPase by altering the lipid environment in its vicinity or by removing an inhibitory subunit.The plant PM2 H+-ATPase is involved in a number of crucial plant functions including acidification of cell walls (which facilitates cell elongation), regulation of cytoplasmic pH, and maintenance of the protonmotive force (which is necessary for the membrane transport of ions and sugars) (24,26,28). In vivo, plant cell wall acidification is stimulated by the plant growth hormone auxin and by the fungal toxin fusicoccin, suggesting that the PM H+-ATPase is under some type of hormonal regulation (20), but the mechanisms remain obscure.ATPase action has frequently been studied using vesicles prepared by sucrose or dextran density-gradient centrifugation (5,7,27,28). This method yields sealed, inside-out PM vesicles in which access of ATP to the ATPase is not a problem, and in

The total lipid composition of plasma membranes (PM), isolated by the phase partitioning method from two different oat (Avena sativa L.) tissues, the root and coleoptfle, was compared. In general, the PM lipid composition was not conserved between these two organs of the oat seedling. Oat roots contained 50 mole percent phospholipid, 25 mole percent glycolipid, and 25 mole percent free sterol, whereas comparable amounts in the coleoptile were 42, 39, and 19 mole percent, respectively. Individual lipid components within each lipid class also showed large variations between the two tissues. Maximum specific ATPase activity in the root PM was more than double the activity in the coleoptile. Treatment of coleoptile with auxin for 1 hour resulted in no detectable changes in PM lipids or extractable ATPase activity. Differences in the PM lipid composition between the two tissues that may define the limits of ATPase activity are discussed.The plant PM2 is a critical component ofthe cell, separating the cytoplasm from the apoplasm. It contains proteins involved in the selective transfer of ions and molecules across this membrane. One important protein is the H+-ATPase, whose transport of protons to the apoplast is important both for the subsequent uptake of sugars and amino acids by proton-organic cotransport and as a component of growth regulation (28). The activity of this enzyme, as well as other PM proteins, is influenced by the composition of the lipid phase in which they are embedded. This influence is indicated both by experiments where proteins are reconstituted into synthetic bilayers of differing lipid composition (8,17,29) and by experiments where lipid components are partially removed by surfactants (24).

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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).

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