Evaluation of Periodate/Lysine/Paraformaldehyde Fixation as a Method for Cross-Linking Plasma Membrane Glycoproteins

Abstract

Fixation by periodate/lysine/paraformaldehyde, a method purported to cross-link specifically plasma membrane glycoproteins, was evaluated using Novikoff rat ascites hepatocellular carcinoma cells. Cells were treated with periodate/lysine, periodate/glycine, and periodate/lysine/paraformaldehyde and subsequently reduced with NaB3H4. The glycoproteins labeled with 3H were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and visualized by fluorography. The effects of reactant concentrations on 3H-labeling of cellular components, cell viability, and cross-linkage of 3H-labeled proteins were examined. The effect of increasing the localized density of plasma membrane glycoproteins on the extent of cross-linkage by periodate and lysine was investigated using cells in which patching of the plasma membrane glycoproteins had been induced by ferritin-conjugated concanavalin A/rabbit antiferritin antiserum. Also investigated was the periodate-independent decrease in pH following addition of paraformaldehyde to mixtures of periodate and lysine or glycine. Results of these studies did not support a mechanism of cross-linking involving reaction between the free base lysine and aldehyde groups on periodate oxidized carbohydrate residues but suggested a complex interaction between periodate oxidized plasma membrane glycoproteins and polymeric complexes of lysine and formaldehyde.