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 u sing anti-chitin synthase IgG. Mild fixation with a mixture of 0.25% g lutaraldehyde and 4% formaldehyde did not destroy the antibody-binding capacity of chitin synthase and preserved the ultrastructure of organ elles. Infusion with a mixture of polyvinylpyrrolidone and sucrose as a cryoprotectant before freezing provided adequate sectioning plastici ty. Immunolabeling of colloidal gold was detected both at the plasma m embrane and the vesicles, and more so in the latter. The results of im munoelectron microscopy were consistent with the fractionation profile of isopycnic sedimentation of the microsomal fraction on a linear suc rose gradient. The profile also demonstrated two peaks of chitin synth ase, 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.