Investigation of the Uptake of Asialo-Glycoproteins by Isolated Rat Hepatocytes. Implications for its Biological Significance

The uptake of asialo-glycoproteins, asialo-fetuin and asialo-orosomucoid, was studied in isolated rat hepatocytes. The total binding capacity for either of these asialo-glycoproteins was 150 000 molecules per cell. The association constants were 0.98 x 10(8) M-1 (asialo-fetuin) and 4.8 x 10(8) M-1 (asialo-orosomucoid) at 10 degrees C. At 37 degrees C, the affinity of asialo-transferrin for the receptor was approximately 1/300 of that of asialo-fetuin. The uptake process was sensitive to neuraminidase, the effect being measurable at 0.5 mU/ml. At 10 mU/ml, the uptake was 15% of that in control cells. The uptake was at the same level when the cells were washed to remove neuraminidase before the addition of asialo-glycoprotein. Lowering the concentration of calcium ions below 2 mM reduced the affinity of the receptor, as addition of an excess of EGTA led to rapid release of asialo-glycoprotein from the cells. The fraction which was released was low when the cells had been incubated with asialo-glycoprotein at 37 degrees C before the addition of EGTA; nearly all of the cell-associated asialo-glycoprotein could be released if the cells had been incubated at 10 degrees C. At 10 degrees C as well as 37 degrees C, the uptake declined sharply when the concentration of calcium ions in the medium was lowered from 0.5 mM to 0.05 mM.