Identification and characterization of lipids from endosomes purified by electromagnetic chromatography.
Traditional separation techniques do not yield endolysosomes of sufficient purity to permit detailed biochemical characterization of this important class of intracellular vesicles. Here, we have used a magnetic chromatography technique to isolate the endosomes from rat peritoneal macrophages and studied their lipid composition. Electromagnetic isolation works by retention of colloidal iron containing vesicles on magnetic column. The data suggested that both early and late endosomes were rich in cholesterol, whereas sphingomyelin (SM) and specific phospholipids like phosphatidylcholine. phosphatidylethanolamine, phosphatidylglycerol and phosphatidylserine are enriched in the late compartments. Our results also indicated that the purified fractions are enriched in raft lipids like SM, but not in cholesterol. The endosomal purification method described here yields pure endosomes with little or no contamination from mitochondria and hence could be used for further biochemical and marker analysis, giving insight into mechanisms of endocytic traffic.
Guha
Sonia
S
Department of Biotechnology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S. G. Highway, Thaltej, Ahmedabad 380054, India.
Rajani
M
M
Padh
Harish
H
D000818:Animals; D002845:Chromatography; D002855:Chromatography, Thin Layer; D055590:Electromagnetic Phenomena; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D008055:Lipids / Q000032:analysis / Q000302:isolation & purification*; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D008055:Lipids