Macromolecule-Macromolecule Interaction in Drug Distribution. II. Effect of [alpha]-Glubulin on Saturable Uptake of Fractionated [3H]Heparin by Rat Parenchymal Hepatocytes in Primary Culture

The uptake of fractionated [3H]heparin was investigated in rat parenchymal hepatocytes in primary culture. The initial uptake of fractionated [3H]heparin was found to be saturable with the maximum uptake velocity (Vmax) of 10.1±1.46 pmol/min/mg protein and the Michaelis constant (Km) of 284±47.9 nM. The effect of α-globulin, the major protein binding to fractionated [3H]heparin, on the saturable uptake profile of fractionated [3H]heparin was also investigated. The uptake clearance was reduced, depending on the concentration of fractionated [3H]heparin, by the addition of 1 mg/ml α-globulin. We assumed that fractionated 3H-heparin bound to α-globulin was not available for uptake and that the reduction in the uptake clearance was solely attributable to the saturable binding of fractionated [3H]heparin to α-globulin. The uptake clearance versus concentration profile was analyzed to obtain the dissociation constant (Kd) of 31.8nM and the capacity (n) of 0.047 for the binding of fractionated [3H]heparin to α-globulin. The saturable binding of fractionated [3H]heparin to α-globulin was supported by in vitro binding experiments using gel chromatography, in which bound fractionated [3H]heparin decreased with the concentration of fractionated [3H]heparin in the presence of α-globulin. In conclusion, the present study demonstrated the saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes and the saturable binding of fractionated [3H]heparin to α-globulin. The saturable uptake may suggest the involvement of a specific transport system such as receptor-mediated endocytosis.