Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting
gluconeogenic enzyme and in liver occurs in a lobular gradient
from periportal to pericentral regions. The subcellular
distribution of cytoplasmic PEPCK molecules within hepatocytes
and its relationship to organelles have not been determined
previously. In this study, we have used immunogold electron
microscopy to evaluate the subcellar distribution of the enzyme,
in addition to brightfield and epipolarized light microscopy.
Cryosections (10 μm) of perfusion-fixed rat liver were
collected on silanated slides and immunostained using goat anti-rat
PEPCK followed by 5-nm gold-labeled secondary and tertiary
antibodies. Additionally, free-floating vibratome sections (25,
50, and 100 μm) of perfusion-immersion-fixed rat liver were
immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled
secondary antibody, with and without silver enhancement. The
immunogold labeled sections from both procedures were embedded
in epoxy resin for the preparation of thin sections for electron
microscopy. The results showed that the gold-labeled antibodies
penetrated the entire thickness of cryosections, resulting in
a high signal for PEPCK, but membranes in general, the smooth
endoplasmic reticulum in particular, were not identifiable as
electron dense unit membranes. On the other hand, the vibratome
sections of well-fixed tissue allowed good visualization of
the ultrastructure of cellular organelles, with the smooth
endoplasmic reticulum appearing as vesicles and tubules with
electron dense unit membranes; however, the penetration of the
gold-labeled antibody was limited to cells at the surface of
the vibratome sections. In both procedures, PEPCK, as indicated
by gold particles, is predominantly in the glycogen areas of
the cytosome and not in mitochondria, nuclei, Golgi apparatus,
or other cell organelles. Hepatocytes in periportal regions
have a compact subcellular distribution of PEPCK shown by gold
particles; hepatocytes in pericentral regions have a diffuse
subcellular distribution of PEPCK and thus more scattered gold
particles. When normal serum replaced the first antibody in
the immunogold staining procedures, the background was very
low.
Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.
