Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H<sup>+</sup>-ATPase Activity and H<sup>+</sup>-Transport
Plasma membrane H<sup>+</sup>-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H<sup>+</sup>-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and...
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MDPI AG
2021-12-01
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| Series: | Biomolecules |
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| author | Nikita K. Lapshin Michail S. Piotrovskii Marina S. Trofimova |
| author_facet | Nikita K. Lapshin Michail S. Piotrovskii Marina S. Trofimova |
| author_sort | Nikita K. Lapshin |
| collection | DOAJ |
| description | Plasma membrane H<sup>+</sup>-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H<sup>+</sup>-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H<sup>+</sup>-ATPase activity. The rate of ATP-dependent ∆µH<sup>+</sup> generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H<sup>+</sup>-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H<sup>+</sup>-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H<sup>+</sup>-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H<sup>+</sup>-transport to sterols in the lipid environment of plasma membrane H<sup>+</sup>-ATPase are discussed. |
| first_indexed | 2024-03-10T04:32:40Z |
| format | Article |
| id | doaj.art-4915dfd433874874be7d84a89160c175 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-10T04:32:40Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-4915dfd433874874be7d84a89160c1752023-11-23T04:00:49ZengMDPI AGBiomolecules2218-273X2021-12-011112189110.3390/biom11121891Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H<sup>+</sup>-ATPase Activity and H<sup>+</sup>-TransportNikita K. Lapshin0Michail S. Piotrovskii1Marina S. Trofimova2K.A. Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPP RAS), 35 Botanicheskaya St., 127276 Moscow, RussiaK.A. Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPP RAS), 35 Botanicheskaya St., 127276 Moscow, RussiaK.A. Timiryazev Institute of Plant Physiology of the Russian Academy of Sciences (IPP RAS), 35 Botanicheskaya St., 127276 Moscow, RussiaPlasma membrane H<sup>+</sup>-ATPase is known to be detected in detergent-resistant sterol-enriched fractions, also called “raft” domains. Studies on H<sup>+</sup>-ATPase reconstituted in artificial or native membrane vesicles have shown both sterol-mediated stimulations and inhibitions of its activity. Here, using sealed isolated plasma membrane vesicles, we investigated the effects of sterol depletion in the presence of methyl-β-cyclodextrin (MβCD) on H<sup>+</sup>-ATPase activity. The rate of ATP-dependent ∆µH<sup>+</sup> generation and the kinetic parameters of ATP hydrolysis were evaluated. We show that the relative sterols content in membrane vesicles decreased gradually after treatment with MβCD and reached approximately 40% of their initial level in 30 mM probe solution. However, changes in the hydrolytic and H<sup>+</sup>-transport activities of the enzyme were nonlinear. The extraction of up to 20% of the initial sterols was accompanied by strong stimulation of ATP-dependent H<sup>+</sup>-transport in comparison with the hydrolytic activity of enzymes. Further sterol depletion led to a significant inhibition of active proton transport with an increase in passive H<sup>+</sup>-leakage. The solubilization of control and sterol-depleted vesicles in the presence of dodecyl maltoside negated the differences in the kinetics parameters of ATP hydrolysis, and all samples demonstrated maximal hydrolytic activities. The mechanisms behind the sensitivity of ATP-dependent H<sup>+</sup>-transport to sterols in the lipid environment of plasma membrane H<sup>+</sup>-ATPase are discussed.https://www.mdpi.com/2218-273X/11/12/1891<i>Pisum sativum</i> L.plasma membrane vesiclessterolsMβCDP-type H<sup>+</sup>-ATPaseH<sup>+</sup>-transport |
| spellingShingle | Nikita K. Lapshin Michail S. Piotrovskii Marina S. Trofimova Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H<sup>+</sup>-ATPase Activity and H<sup>+</sup>-Transport Biomolecules <i>Pisum sativum</i> L. plasma membrane vesicles sterols MβCD P-type H<sup>+</sup>-ATPase H<sup>+</sup>-transport |
| title | Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H<sup>+</sup>-ATPase Activity and H<sup>+</sup>-Transport |
| title_full | Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H<sup>+</sup>-ATPase Activity and H<sup>+</sup>-Transport |
| title_fullStr | Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H<sup>+</sup>-ATPase Activity and H<sup>+</sup>-Transport |
| title_full_unstemmed | Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H<sup>+</sup>-ATPase Activity and H<sup>+</sup>-Transport |
| title_short | Sterol Extraction from Isolated Plant Plasma Membrane Vesicles Affects H<sup>+</sup>-ATPase Activity and H<sup>+</sup>-Transport |
| title_sort | sterol extraction from isolated plant plasma membrane vesicles affects h sup sup atpase activity and h sup sup transport |
| topic | <i>Pisum sativum</i> L. plasma membrane vesicles sterols MβCD P-type H<sup>+</sup>-ATPase H<sup>+</sup>-transport |
| url | https://www.mdpi.com/2218-273X/11/12/1891 |
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