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Purification of plasma membrane from Acanthamoeba castellanii
B J Clarke1, T C Hohman, B Bowers
1Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Abstract:
A simple method for isolation of plasma membrane from Acanthamoeba using self-generating gradients of Percoll is described. To obtain a membrane marker, intact amoebae were radioiodinated and the distribution of the radiolabel was followed through the plasma membrane isolation procedure. The purity of isolated plasma membrane was assessed by enrichment of radiolabel, by electron microscopy, and by enzymatic assays for contaminating membranes. As judged from enrichment of radiolabel, a 37-fold purification of plasma membrane was obtained. We estimate that 80% of the total protein was from plasma membrane and 10% from membrane-associated actin.
Insights
Researchers developed a simple method to isolate plasma membranes from Acanthamoeba using Percoll gradients. This technique achieved a 37-fold purification, yielding highly pure plasma membrane for further study.
Area of Science:
- Cell Biology
- Biochemistry
- Microbiology
Background:
- Isolation of pure plasma membrane is crucial for understanding cellular functions.
- Acanthamoeba serves as a model organism for studying protozoan biology.
Purpose of the Study:
- To develop a simple and efficient method for isolating plasma membrane from Acanthamoeba.
- To assess the purity and yield of the isolated plasma membrane.
Main Methods:
- Utilized self-generating gradients of Percoll for plasma membrane isolation.
- Employed radioiodination of intact amoebae to trace membrane distribution.
- Assessed purity using radiolabel enrichment, electron microscopy, and enzymatic assays.
Main Results:
- Achieved a 37-fold purification of Acanthamoeba plasma membrane.
- Estimated 80% of the isolated protein originated from the plasma membrane.
- Identified 10% of the protein as membrane-associated actin.
Conclusions:
- The described Percoll gradient method is effective for isolating highly pure Acanthamoeba plasma membrane.
- This method provides a valuable tool for biochemical and cell biological studies of Acanthamoeba.
- The isolated membrane fraction is suitable for investigating plasma membrane composition and function.