Detection of rat ceruloplasmin receptors using fluorescence microscopy and microdensitometry

Rat ceruloplasmin (rCp) has been labeled with the fluorophores fluorescein and rhodamine by using the isothiocyanate derivatives (FITC, RBITC). High p-phenylenediamine oxidase activity of the resulting conjugates was observed (70-90% of native activity). Polyacrylamide gel electrophoresis showed fluorescein-labeled rCp (FITC-rCp) had an increased mobility, while rhodamine-labeled rCp (RBITC-rCp) showed no increase in mobility when compared to native rCp. RBITC-rCp was tested as a probe for ceruloplasmin receptors on rat erythrocytes using fluorescence microscopy to detect membrane binding. Film negatives of blood smears exposed under identical conditions were analyzed by microdensitometry to give relative optical densities for the amount of RBITC-rCp bound per unit area of the plasma membrane. With this technique, binding of rCp was observed to be saturable, reversible, and specific. Competition of the protein ligands superoxide dismutase, catalase, and unlabeled rCp against RBITC-rCp already bound on erythrocytes showed that only rCp could displace the bound RBITC-rCp with 32% specific binding being observed. Low levels of membrane binding were seen after the erythrocytes had been trypsin treated. Platelet binding was also detected and it was saturable, reversible, and trypsin sensitive. However, only 20% of the bound RBITC-rCp could be displaced by rCp. These studies demonstrate a versatile technique for detection and localization of Cp receptors.