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Abstract <p>The kinetic parameters of ATP hydrolysis in plasma membrane fractions of pea root cells (<i>Pisum sativum</i> L.) with different densities were studied. The plasma membrane obtained by separation of microsomal membranes in the aqueous two-phase polymer system was fractionated in the discontinuous iodixanol density gradient into light (≤1.15 g/cm<sup>3</sup>) and heavy (≥1.15 g/cm<sup>3</sup>) fractions with their further analysis for the H<sup>+</sup>-ATPase and sterol content. Then, the lipid packing of the membrane bilayer and hydrolytic activity of the H<sup>+</sup>-ATPase in these fractions were analyzed. It was found that the light fraction was characterized by an increased amount of both sterols and H<sup>+</sup>-ATPase compared to the heavy sample, as well as possessed high affinity for ATP. However, the sterol-depleted heavy fraction showed a high rate of ATP hydrolysis (<i>V</i><sub>max</sub>) simultaneously with an increased <i>K</i><sub><i>M</i></sub> value. Experiments with the fluorescent dye Laurdan showed that the fractions were slightly differed in the lipid packing of the membrane bilayer. Two possible mechanisms behind the modulation of H<sup>+</sup>-ATPase activity by membrane sterols are discussed.</p>

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Mechanisms of Plant Plasma Membrane H+-ATPase Regulation Mediated by Its Interaction with Membrane Sterols

  • 期刊论文

  • 发表日期：

    2025年7月1日

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  • N. K. Lapshin,
  • M. S. Piotrovskii,
  • M. S. Trofimova

摘要

Abstract

The kinetic parameters of ATP hydrolysis in plasma membrane fractions of pea root cells (Pisum sativum L.) with different densities were studied. The plasma membrane obtained by separation of microsomal membranes in the aqueous two-phase polymer system was fractionated in the discontinuous iodixanol density gradient into light (≤1.15 g/cm3) and heavy (≥1.15 g/cm3) fractions with their further analysis for the H+-ATPase and sterol content. Then, the lipid packing of the membrane bilayer and hydrolytic activity of the H+-ATPase in these fractions were analyzed. It was found that the light fraction was characterized by an increased amount of both sterols and H+-ATPase compared to the heavy sample, as well as possessed high affinity for ATP. However, the sterol-depleted heavy fraction showed a high rate of ATP hydrolysis (Vmax) simultaneously with an increased KM value. Experiments with the fluorescent dye Laurdan showed that the fractions were slightly differed in the lipid packing of the membrane bilayer. Two possible mechanisms behind the modulation of H+-ATPase activity by membrane sterols are discussed.