Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells

Abstract

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (beta-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X(7) receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.

Clustering endothelial E-selectin in clathrin-coated pits and lipid rafts enhances leukocyte adhesion under flow

“…on April 30, 2019. by guest www.bloodjournal.org From of cells extracted with or without detergent, whereas some markers fractionate very differently depending on the extraction method. 50 The clathrin-dependent endocytosis of E-selectin (Figure 1) and the colocalization of a portion of E-selectin with the clathrin-pit marker ␣-adaptin (Figure 2) strongly suggest that not all E-selectin is in lipid rafts. We also used confocal microscopy to compare the colocalization of E-selectin with caveolin-1, GM1, and flotillin-1 in CHO cells and HUVECs.…”

Interaction and interrelation of P2X7 and P2X4 receptor complexes in mouse lung epithelial cells

“…DRMs (lipid rafts) are sphingolipid-and cholesterolrich membrane microdomains that are involved in receptor signaling, vesicle traffic and protein sorting [21,22]. The localization in DRMs was also reported for P2X3R in neurons and for P2X1R in smooth muscle cells [23,24], as well as for P2X7R in mouse T cells, rat submandibular gland and macrophages [25][26][27]. Caveolae are specialized microdomains in the ATI cells, which form flask-like invaginations of the plasma membrane [22].…”

Biochemical characterization of membrane fractions in murine sperm: Identification of three distinct sub‐types of membrane rafts

“…A discontinuous sucrose gradient of 5%/35%/45% has been widely used to partition the membrane domains, with rafts being collected at the interface between 5% and 35% (Garcia-Marcos et al, 2006a; Garcia-Marcos et al, 2006b). However, significant contamination with non-raft proteins can occur using this methodology (Foster et al, 2003).…”