FITC-POLY-D-LYSINE CONJUGATES AS FLUORESCENT-PROBES TO QUANTIFY HAPTEN-SPECIFIC MACROPHAGE RECEPTOR-BINDING AND UPTAKE KINETICS

A series of fluorescein derivatized poly-D-lysine (FITC-PDL) probes we re used to elucidate the role of fluorescein in receptor binding of fl uorescein-conjugated macromolecules to J774 murine macrophages, Poly-D -lysine served to eliminate receptor recognition of the carrier due to the biologically inert nature of the D-isomer, This concept enabled t he focused investigation of the role played by fluorescein in receptor recognition, binding and internalization. Results revealed dependency of cellular uptake on polymer concentration, hapten density and acces sibility, The results differed from those previously obtained with FIT C-BSA in that saturating fluorescein densities on the poly-D-lysine po lymer resulted in diminished rates of uptake by macrophages, Receptor- mediated endocytosis via clathrin-coated pits was concluded based on r esults that showed inhibition of FITC-PDL uptake by intracellular K+ d epletion but not by the macropinocytosis inhibitor, amiloride, Further , FITC-PDL was found to inhibit the endocytic uptake of FITC-BSA sugge sting competition between the two probes at the level of a macrophage receptor Association rates (k(on)) for binding to the macorphage surfa ce were measured for the various FLTC-PDL probes based on fractional r eceptor occupancies, Results are discussed on the basis of receptor re cognition of fluorescein in J774 macrophages and the requirements for this recognition which include appropriate spacing and accessibility o f the hapten moieties to facilitate receptor crosslinking. (C) 1998 Wi ley-Liss, Inc.