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Isolation and characterization of subcellular membranes of Entamoeba invadens
Abstract:
A method is described for the isolation of subcellular membranes of Entamoeba invadens. Plasma membranes were obtained by rate centrifugation followed by isopycnic centrifugation on a sucrose gradient. Intact phagolysosomes floated in a 10% sucrose solution providing a simple technique for isolation. Phagolysosomal membranes were collected by isopycnic centrifugation, after lysis of the phagolysosomes. Microsomes were obtained by differential centrifugation. Membrane fractions were examined by electron microscopy, and the contamination of each fraction was determined with marker enzymes. Mg2+-ATPase is associated with the plasma membrane. Acid phosphatase (beta-glycerophosphate) was associated mainly with phagolysosmal membranes. Plasma membranes also contained acid phosphatase activity which hydrolyzes p-nitrophenylphosphate but not beta-glycerophosphate. The localization of the two phosphatases was confirmed cytochemically. Isolated plasma membranes were contaminated with phagolysosomal membranes (15%) and with microsomes (25%). No more than 5% of the phagolysosomal membrane fraction consisted of plasma membranes. Contamination of the microsomes by plasma and phagolysosomal membranes was 10% and 7%, respectively. Plasma membranes and phagolysosomal membranes had a high ratio of cholesterol to phospholipid (0.93 and 1.05 mumol/mumol, respectively). Microsomes were relatively poor in cholesterol (0.39 mumol/mumol). Microsomes, plasma, and phagolysosomal membranes contained increasing amounts of spingolipids (12%, 17%, and 28%). Phagolysosomal membranes had a high percentage of phosphatidylserine but little phosphatidylcholine. Microsomes were rich in phosphatidylcholine (45%). Differences in phospholipid composition between plasma and phagolysosomal membranes are discussed in view of the phagocytic process.
Insights
Researchers developed a method to isolate cellular membranes from Entamoeba invadens. This technique successfully separated plasma membranes, phagolysosomes, and microsomes, revealing distinct lipid compositions crucial for understanding phagocytosis.
Area of Science:
- Cell Biology
- Biochemistry
- Parasitology
Background:
- Understanding the distinct functions of cellular membranes is crucial for comprehending cellular processes like phagocytosis.
- Entamoeba invadens serves as a model organism for studying parasitic protozoa and their cellular mechanisms.
Purpose of the Study:
- To develop and validate a method for isolating specific subcellular membrane fractions from Entamoeba invadens.
- To characterize the biochemical and lipid composition of isolated plasma membranes, phagolysosomal membranes, and microsomes.
Main Methods:
- Isolation of subcellular membranes using a combination of rate zonal and isopycnic centrifugation techniques.
- Characterization of membrane fractions through electron microscopy and marker enzyme analysis (Mg2+-ATPase, acid phosphatase).
- Analysis of lipid composition, including cholesterol, phospholipid, and sphingolipid content.
Main Results:
- Successfully isolated plasma membranes, phagolysosomes, and microsomes with defined contamination levels.
- Identified specific enzyme markers for each membrane fraction (Mg2+-ATPase for plasma membranes, acid phosphatase for phagolysosomal membranes).
- Revealed significant differences in cholesterol, phospholipid, and sphingolipid profiles across the membrane fractions, particularly between plasma and phagolysosomal membranes.
Conclusions:
- The described method provides a reliable approach for isolating distinct membrane fractions from Entamoeba invadens.
- The distinct lipid compositions suggest specialized roles for plasma and phagolysosomal membranes in the phagocytic process.
- Further investigation into these membrane differences can elucidate the mechanisms of phagocytosis in parasitic protozoa.