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Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei
Abstract:
The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.
Insights
Phospholipase A treatment of Mycobacterium phlei membranes affects electron transport and phosphorylation. Phosphorylation can occur without a significant pH gradient, suggesting altered membrane hydrophobicity.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Electron transport particles and coupling factor-depleted particles from Mycobacterium phlei share similar phospholipid composition but differ in content.
- The accessibility of phospholipase A to membrane phospholipids varies between these particle types.
Purpose of the Study:
- To investigate the role of phospholipids in electron transport and phosphorylation in Mycobacterium phlei.
- To determine the effect of phospholipase A treatment on membrane properties and energy coupling.
Main Methods:
- Treatment of Mycobacterium phlei membranes with phospholipase A.
- Assay of oxidation and phosphorylation rates.
- Reconstitution of coupling factor-latent ATPase activity.
- Measurement of pH gradient using bromthymol blue.
- Fluorescence studies using 1-anilino-8-naphthalene sulfonic acid (ANS).
Main Results:
- Phospholipase A treatment impaired oxidation and phosphorylation, with phosphorylation inhibition being reversible by bovine serum albumin.
- Reconstitution of ATPase activity and substrate phosphorylation coupling were unaffected by phospholipase A, but pH gradient restoration failed.
- ANS fluorescence decreased in phospholipase A-treated membranes, correlating with phospholipid cleavage and reduced membrane hydrophobicity.
Conclusions:
- Phosphorylation coupled to substrate oxidation can occur in Mycobacterium phlei membrane vesicles independently of a pronounced pH gradient.
- Alterations in membrane phospholipid composition, specifically decreased hydrophobicity, affect energy-dependent ANS fluorescence.
- These findings highlight the complex interplay between membrane structure, phospholipid content, and energy transduction in Mycobacterium phlei.