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Transport properties of membrane vesicles from Acholeplasma laidlawii. I. Isolation and general characteristics
Abstract:
A new procedure for the isolation of membrane vesicles from Acholeplasma laidlawii cells is described. The membrane vesicles are completely free from contaminations of whole cells and cell debris and represent a homogeneous fraction as shown by electron microscopy, Ficoll density-gradient centrifugation, and titration on agar plate. Absence of cytoplasmic contaminations was confirmed by double-labelling of membranes with 3H-oleic acid and 14C-uridine, as well as by distribution of specific marker enzymes of membranes and cytoplasm. On the basis of light-scattering and electron microscopy, the vesicular nature of these membranes was established. The vesicles had the same orientation as intact cells (absence on membrane vesicles of ATPase and NADH dehydrogenase activities, localized in the inner surface of membrane). The respiratory activity of the membrane vesicles was low and was not stimulated by exogenous substrates, the respiratory chain of the vesicles being reduced and terminated by flavoproteins. The ability of membrane vesicles to take up carbohydrates was shown.
Insights
Researchers developed a novel method to isolate pure membrane vesicles from Acholeplasma laidlawii. These vesicles, free from cellular contaminants, retain their original orientation and exhibit carbohydrate uptake capabilities.
Area of Science:
- Microbiology
- Cell Biology
- Biochemistry
Background:
- Acholeplasma laidlawii is a unique bacterium lacking a cell wall.
- Understanding its membrane structure and function is crucial for cell biology research.
- Isolation of pure membrane vesicles is challenging due to potential contaminants.
Purpose of the Study:
- To develop and validate a new procedure for isolating pure membrane vesicles from Acholeplasma laidlawii.
- To characterize the purity, homogeneity, and orientation of the isolated membrane vesicles.
- To investigate the functional properties of these membrane vesicles, including respiratory activity and substrate uptake.
Main Methods:
- Isolation of membrane vesicles using a novel procedure.
- Purity assessment via electron microscopy, Ficoll density-gradient centrifugation, and agar plate titration.
- Cytoplasmic contamination analysis using double-labeling (3H-oleic acid, 14C-uridine) and marker enzyme distribution.
- Vesicle characterization through light-scattering and electron microscopy.
- Enzyme activity assays for ATPase and NADH dehydrogenase.
- Measurement of respiratory activity and carbohydrate uptake.
Main Results:
- A highly pure and homogeneous fraction of membrane vesicles was obtained, free from whole cells and debris.
- Absence of cytoplasmic contamination was confirmed by multiple methods.
- Vesicles were shown to possess the same orientation as intact cells, with specific enzyme activities localized correctly.
- Low respiratory activity was observed, indicating a reduced respiratory chain terminated by flavoproteins.
- Demonstrated ability of membrane vesicles to take up carbohydrates.
Conclusions:
- The described procedure effectively isolates pure, uniformly oriented membrane vesicles from Acholeplasma laidlawii.
- These vesicles are suitable for studying membrane-bound enzymes and transport systems.
- The findings provide insights into the functional properties of Acholeplasma laidlawii membranes.