Cytochemical detection of mannose-specific receptors for glycoproteins with horseradish peroxidase as a ligand.
Horseradish peroxidase (HRP), a glycoprotein rich in mannose groups, was used as a ligand to detect receptors for glycoproteins in formalin-fixed, frozen sections of rat liver. Specific binding of HRP occurred to surface membranes of sinusoidal cells but not to those of parenchymal cells. The binding sites were visualized after the peroxidatic reaction in erythrocytes had been suppressed by methanol-H2O2 and phenylhydrazine, the latter reagent also decreasing the nonspecific background adsorption of HRP. Several factors influencing the reaction were studied systematically. The specific binding of HRP to sinusoidal cells was greatly decreased or abolished when tissue blocks were fixed for longer than 1-2 h in a cold 4% formaldehyde solution and the frozen sections subsequently treated for 30 min in cold methanol. The specific binding of HRP increased when the concentration of HRP in the medium was increased from 10 microgram/ml to 40 microgram/ml, when the time of incubation with HRP was increased from 1 h to 4 h, or when the temperature of incubation with HRP was increased from 4 degrees C to 22 degrees C, the pH of the incubation medium was increased from 7.0 to 10.0. Little or no specific binding of HRP was observed in the absence of added Ca++. The binding of HRP was suppressed by 10 mM mannose or 0.004% mannan whereas the suppression of the binding reaction by galactose or galactan required 30-40 ties higher concentrations.
D000818:Animals; D001665:Binding Sites; D006023:Glycoproteins / Q000378:metabolism*; D006651:Histocytochemistry; D006735:Horseradish Peroxidase; D008099:Liver / Q000378:metabolism; D008358:Mannose / Q000378:metabolism*; D010544:Peroxidases; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*
D006023:Glycoproteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; C032193:glycoprotein receptor GPIb-IX; D006735:Horseradish Peroxidase; D010544:Peroxidases; D008358:Mannose