Use of monoclonal antibody and colloidal gold in E.M. localization of von Willebrand factor in megakaryocytes and platelets

The subcellular localization of Factor VIII/von Willebrand protein (VIII R:Ag) is studied with monoclonal antibody and gold immunocytochemical technique. Monoclonal antibody against purified VIII R:Ag is brightly fluorescent on megakaryocytes and platelets. In E.M., gold immunolabeling is performed on thin cell sections of human megakaryocytes and platelets. Different embedding materials are used to preserve the antigenicity : Epon embedded megakaryocytes show a high concentration of VIII R:Ag in alpha-granules using 4F9 monoclonal antibody. In comparison, lowicryl K4M embedded material does not improve the same specificity, only a few platelets granules were stained. This subcellular localization, in full agreement with biochemical results appears visualized for the first time in E.M.     

Processing of snake venom -amino acid oxidase during intracellular transport

The isoelectrofocusing patterns of -amino acid oxidase (LAO) from venom gland homogenates and of the secreted venom of Vipera palaestinae have been compared. The LAO isozyme profile of actively synthesizing gland spans over a wider range of pIs (4.8–6.0) and includes more variants as compared with the profile of the secreted venom. A basic shift of the isoelectrofocusing pattern of LAO obtained by treatment of the gland homogenate or the venom with neuraminidase indicates that sialic acid residues are responsible for the changes in the electronegativity of the isozymes. Analyses of subcellular fractions show that the microsomal fraction of the venom gland homogenate exhibits the highest multiplicity of molecular forms of LAO, whereas the fraction including the secretory granules has an isozyme profile similar to the venom. Double labelling experiments show that the newly synthesized LAO include isozymes which span over a wide range of pIs, whereas later on labelling of the more acidic isozymes is prominent. The results obtained may suggest that the sialic acid residues which are attached to LAO during its transport serve as “markers” for secretion.