Study of surface carbohydrates on isolated Golgi subfractions by fluorescent-lectin binding and flow cytometry.
The Golgi complex is a functionally heterogeneous subcellular structure that plays a key role in the synthesis, maturation, and sorting of newly synthesized glycoproteins. Fluorescent lectins have been used extensively to analyze surface glycoproteins by flow cytometry in whole cells and more recently in isolated subcellular organelles, such as mitochondria and chloroplasts. We report here the use of several fluorescein-isothiocyanate-conjugated lectins to detect and quantify specific surface sugars by flow cytometry on isolated elements from purified cis and trans-Golgi fractions from rat liver. Our results show that this approach may be useful to study Golgi composition and function, since it may reveal the intensity of specific binding of different lectins to each Golgi fraction and the percentage of elements binding the lectins specifically. Thus we show here that Golgi elements appear homogeneous in mannose and fucose, whereas galactose and N-acetyl-glucosamine residues are more abundant in the trans-Golgi elements.
Guasch
R M
RM
Departamento de Bioquímica y Biología Molecular (Centro de Referencia Coulter-Izasa S.A.), Facultad de Medicina, Universidad de Valencia, Spain.
Guerri
C
C
O'Connor
J E
JE
D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D005434:Flow Cytometry / Q000295:instrumentation; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000737:chemistry*; D007425:Intracellular Membranes / Q000737:chemistry; D017879:Isothiocyanates; D037102:Lectins / Q000378:metabolism*; D008099:Liver / Q000737:chemistry* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000032:analysis; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar
D002241:Carbohydrates; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D037102:Lectins; D008562:Membrane Glycoproteins; C037152:isothiocyanic acid