Steady state and kinetic analysis of the binding of asialoorosomucoid to the isolated rabbit hepatic lectin.
The binding of 125I-asialoorosomucoid and the isolated rabbit hepatic lectin were studied at 0 degrees C. The steady state binding data were analyzed both by a direct curve-fitting procedure that utilizes nonlinear least squares regression analysis, and by conventional Scatchard plot analysis. Two classes of binding sites, present in approximately equal concentrations, were detected by both analytical procedures. The total amount of 125I-asialoorosomucoid bound was between 0.2 and 1.0 mol/mol of lectin for each class of lectin binding sites, if the molecular weight of the lectin is between 100,000 and 500,000. The estimated apparent equilibrium dissociation constants were Kapp = 0.87 nM and 1100 nM using the direct curve-fitting method and 0.63 nM and 43 nM from Scatchard plot analysis. Analysis of the forward binding reaction yielded apparent rate constants k1 = 6.8 X 10(6) M-1 and k-1 = 1.5 X 10(-2) min-1, and, therefore, Kapp = 2.1 nM. (Kapp is the apparent dissociation constant of the complex between asialoorosomucoid and hepatic lectin, K1 is the bimolecular rate constant for association of asialoorosomucoid and hepatic lectin, and K-1 is the rate constant for dissociation of the asialoorosomucoid-hepatic lectin complex.) In contrast, the reverse reaction was too slow in the presence of excess unlabeled asialoorosomucoid, and too fast in the presence of methyl beta-D-galactopyranoside to be accommodated by the simple bimolecular reaction model.
D000818:Animals; D001212:Asialoglycoproteins; D007700:Kinetics; D037102:Lectins / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D017830:Octoxynol; D009961:Orosomucoid / Q000031:analogs & derivatives* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011817:Rabbits; D013501:Surface-Active Agents / Q000494:pharmacology
D001212:Asialoglycoproteins; D037102:Lectins; D009961:Orosomucoid; D013501:Surface-Active Agents; C017030:asialoorosomucoid; D011092:Polyethylene Glycols; D017830:Octoxynol