Subfractionation of liver membrane preparations by specific ligand-induced density perturbation.

Immunofluorescence and immunoferritin staining with monospecific antibodies to dipeptidyl peptidase IV purified from rat liver plasma membrane showed that the antigenic sites of this glycoprotein was exposed only on the outer surface of the liver cell. In a vesiculated plasma membrane preparation the peptidase was located exclusively on right-side-out elements, which differed in their degrees of ferritin staining, and could be separated into subfractions of different buoyant densities corresponding to their concentration of dipeptidyl peptidase IV. The concomitant density perturbation of nucleotide pyrophosphatase was similar, but not identical, to that of the peptidase itself, indicating that these two marker enzymes are somewhat differently distributed in the plane of the liver plasma membrane. Since essentially all the galactosyl transferase in plasma membrane and none of that in Golgi membrane could be density-perturbed with the antipeptidase, the activity in the plasma membrane preparation could not be ascribed to contamination with discrete Golgi elements. On the other hand, the small amount of dipeptidyl peptidase IV found in the Golgi preparations was itself perturbed by the antipeptidase, indicating that it represented contaminating right-side-out plasma membrane vesicles. In preliminary experiments similar separations were also obtained with wheat germ agglutinin as the plasma membrane ligand. Density perturbation, mediated by the recognition of specific surface markers, should be a useful adjunct in the separation and characterization of subcellular components in other systems.