Quantification of concanavalin A binding to rat brain microsomal membranes detected by fluorescence polarization technique.
A high sensitive method for detecting the change of microsomal membrane surface oligosaccharides was developed to study the regulatory role of lipid- or peptide-linked mannoside of endoplasmic reticulum in synaptic functions. The binding of concanavalin A to the microsomal membrane surface was measured quantitatively using a microgram-order of rat brain microsomal proteins. The fluorescence polarization of concanavalin A (Con A)-fluorescein isothiocyanate (FITC) conjugate bound to the membrane was analyzed to quantitate the change of binding constant and the number of binding sites. As a control, the non-specific binding of bovine serum albumin-FITC conjugate was measured by the same technique. We measured the change of fluorescence intensity of membrane-bound FITC conjugates by the flow cytometry and found that the intensity of FITC conjugate bound to the membrane increased more than that of free form of the probe. We observed that the alpha-mannosidase-treatment of rat brain microsomes resulted in the increase of binding constant of Con A to the microsomal surface without significant loss of binding sites.
Miyazaki
M
M
Research Laboratory Center, Oita Medical University, Hasama-cho, Oita 879-55, Japan.
Yoshida
S
S
Sakai
K
K
Zhang
Q Z
QZ
Takeshita
M
M
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D003208:Concanavalin A / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005454:Fluorescence Polarization; D007425:Intracellular Membranes / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D012680:Sensitivity and Specificity; D043323:alpha-Mannosidase
D008565:Membrane Proteins; D009844:Oligosaccharides; D003208:Concanavalin A; D008361:Mannosidases; D043323:alpha-Mannosidase; D016650:Fluorescein-5-isothiocyanate