We developed a system of techniques--"fracture--label" cytochemistry--where fixed cells or tissues are freeze-fractured, thawed and labeled by conventional cytochemical methods. Labeling of both the protoplasmic nd exoplasmic halves of fractured membranes can establish the composition of fractured membranes and the existence of specific transmembrane proteins. Fracture-label does not involve isolation and fractionation procedures: the membranes are fractured and marked in situ. In columnar, duodenal and globlet cells, we have shown that contrary to expectations, the membranes of the endoplasmic reticulum and the nuclear envelope contain numerous fructose residues labeled by Ulex europaeus I (UEA). As expected, these membranes are not labeled by WGA. We conclude that subterminal implantation of sugars (fructose) does not prevent the backflow of glycoproteins. Numerous UEA receptors are labeled on P faeces what shows that they are in part associated to transmembrane proteins. We have labeled cross-fractured nuclei and shown the association of glycoproteins to enchromatin. These results are the first qualitative, ultrastructural localization of glycoconjugates within the nuclear matrix. Fracture-label of human platelets from normal subsets as well as Bernard-Soulier and Glauzman Thrombasthenia shows that Con A and WGA receptors are associated with components that partition with the outer half of the membrane. In guinea pig mammary gland epithelia cells we show that butyrophilin, a glycoprotein of mile fat globule membranes, is present on the apical plasma membrane as well as on fat droplets inside the cytoplasm. Butyrophilin was detected by labeling with polyclonal antibody followed by protein A colloidal gold. In human neutrophils, choleragenoid/coloidal gold conjugates label E and P faces of the plasma membranes. Label on P faces may reflect the association of GM1 to a transmembrane complex.
NIH ExPorter
NIH ExPorter