Selective delipidation of the plasma membrane by surfactants : enrichment of sterols and activation of ATPase.

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 altering the hydrophobic environment around the enzyme. We propose that sterols, through their interaction with the ATPase, may be essential for ATPase activity.

Comparison of the lipid composition of oat root and coleoptile plasma membranes: lack of short-term change in response to auxin.

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 coleoptile, 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.

Latency of Plasma Membrane H-ATPase in Vesicles Isolated by Aqueous Phase Partitioning : Increased substrate Accessibility or Enzyme Activation.

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 degrees 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.