Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1997 Jul 25;1327(2):222-30.
doi: 10.1016/s0005-2736(97)00061-8.

A biotinylated perfringolysin O derivative: a new probe for detection of cell surface cholesterol

M Iwamoto  1 , I Morita, M Fukuda, S Murota, S Ando, Y Ohno-Iwashita
Affiliations
Free article

A biotinylated perfringolysin O derivative: a new probe for detection of cell surface cholesterol

M Iwamoto et al. Biochim Biophys Acta. .
Free article

Erratum in

  • Biochim Biophys Acta 1998 Mar 6;1370(1):184-5

Abstract

theta-Toxin is a cholesterol-binding, pore-forming cytolysin of Clostridium perfringens. To detect cell surface cholesterol, we prepared a theta-toxin derivative, BC theta by biotinylation of a protease-nicked theta-toxin, which has the same binding affinity for cholesterol as theta-toxin without cytolytic activity. Human erythrocytes, V79 cells and human umbilical vein endothelial cells (HUVEC), were stained with BC theta coupled with FITC-avidin, and then the cells were analyzed by either flow cytometry or laser confocal microscopy. The fluorescence intensity increased in both intact and briefly fixed cells when treated with BC theta. BC theta-treated V79 cells were stained by neither trypan blue nor propidium iodide, indicating that BC stained just the outer surface of the plasma membrane of vital cells. Treatment of the cells with digitonin, a cholesterol-sequestering reagent, decreased the fluorescence intensity to the background level, indicating that BC theta staining is specific for cholesterol. The fluorescence intensity of erythrocytes pre-permeabilized with a small amount of theta-toxin increased more than ten-fold, suggesting higher cholesterol contents in the inner layer of the plasma membrane. When cells were cultured with cholesterol-depleted medium, the fluorescence intensity stained by BC theta decreased remarkably in V79 cells, but did not change in HUVEC. This indicates that cell surface cholesterol may be provided in different ways with these two cell lines. These results suggest that BC theta can be a useful probe for visualizing cell surface cholesterol and for evaluating the effects of cellular events on the topology and distribution of cholesterol.

PubMed Disclaimer

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

LinkOut - more resources