LOCALIZATION OF CHITIN SYNTHASE IN ABSIDIA-GLAUCA STUDIED BY IMMUNOELECTRON MICROSCOPY - APPLICATION OF CRYOULTRAMICROTOMY

Application of cryoultramicrotomy to immunoelectron microscopy showed the localization of the chitin synthase of Absidia glauca in situ by using anti-chitin synthase IgG. Mild fixation with a mixture of 0.25% glutaraldehyde and 4% formaldehyde did not destroy the antibody-binding capacity of chitin synthase and preserved the ultrastructure of organelles. Infusion with a mixture of polyvinylpyrrolidone and sucrose as a cryoprotectant before freezing provided adequate sectioning plasticity. Immunolabeling of colloidal gold was detected both at the plasma membrane and the vesicles, and more so in the latter. The results of immunoelectron microscopy were consistent with the fractionation profile of isopycnic sedimentation of the microsomal fraction on a linear sucrose gradient. The profile also demonstrated two peaks of chitin synthase, one of these cosedimented with vanadate-sensitive ATPase and the other with lower buoyant density populations. The former corresponded to the plasma membrane and the later, vesicles in the cytoplasm.