Latency of Plasma Membrane H⁺-ATPase in Vesicles Isolated by Aqueous Phase Partitioning

Abstract

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

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

“…Extraction of microsomal vesicles, ATPase assays and protein analyses are described in Sandstrom et al (24).…”

“…The procedure has been described in Sandstrom et al (24). Briefly, the microsomal pellet was subjected to three successive phase partitioning steps utilizing 6.5% (w/w) dextran (500,000 mol wt) and polyethylene glycol (3, (19) attached to a 10 mL glass syringe.…”

“…Oats (Avena sativa L. cv Victory) were grown in vermiculite as described previously (24). Briefly, the coleoptiles were grown and harvested after 4 d under continuous dim red light (to inhibit mesocotyl growth) at 25°C.…”

“…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).…”

Calcium and Proton Transport in Membrane Vesicles from Barley Roots

“…The volumes of isolated membrane vesicles were determined using a modification of an ESR technique (1,25,29 A signal-subtraction technique made it possible to use a low concentration of K3Fe(CN)6 that had no significant osmotic effect on the vesicles. A concentration of 33 mM K3Fe(CN)6 broadened the external signal sufficiently to give a signal composed of a superposition of the broadened external and unbroadened internal signal.…”