A photoreactive derivative of radiolabeled GM1 ganglioside: Preparation and use to establish the involvement of specific proteins in GM1 uptake by human fibroblasts in culture

Journal Article · Tue Jan 10 00:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00427a012· OSTI ID:5482341

Sonnino, S; Chigorno, V; Acquotti, D; Pitto, M; Kirschner, G; Tettamanti, G ^[1]

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1. Univ. of Milan (Italy)

A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with ({sup 3}H)acetic anhydride of very high specific radioactivity. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 for different times and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2 h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins. When the incubation was prolonged to 24 h, photolabeled GM1 underwent extensive metabolic processing and gave origins to the corresponding ganglioside derivatives of GM2, GM3, and GD1a. Under these conditions many proteins became radioactive, a consequence of GM1 transfer from the surface to the interior or the cell and of the ready availability of interaction of GM1 and its metabolites.

• OSTI ID: 5482341
• Journal Information: Biochemistry; (USA), Vol. 28:1; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
CELL MEMBRANES
CROSS-LINKING
GANGLIOSIDES
DERIVATIZATION
PROTEINS
AZIDES
BIOELECTRICITY
CELL CULTURES
ELECTROPHORESIS
FIBROBLASTS
HEAVY WATER
LABELLING
MAN
NITRO COMPOUNDS
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
PROTONS
TRITIUM COMPOUNDS
ANIMAL CELLS
ANIMALS
BARYONS
CARBOHYDRATES
CELL CONSTITUENTS
CHEMICAL REACTIONS
CONNECTIVE TISSUE CELLS
ELECTRICITY
ELEMENTARY PARTICLES
FERMIONS
GLYCOLIPIDS
HADRONS
HYDROGEN COMPOUNDS
LIPIDS
MAGNETIC RESONANCE
MAMMALS
MEMBRANES
NITROGEN COMPOUNDS
NUCLEONS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
POLYMERIZATION
PRIMATES
RESONANCE
SACCHARIDES
SOMATIC CELLS
SPECTRA
VERTEBRATES
WATER
550201* - Biochemistry- Tracer Techniques