Localization of glucose-6-phosphate dehydrogenase activity on ribosomes ofgranular endoplasmic reticulum, in peroxisomes and peripheral cytoplasm ofrat liver parenchymal cells
Wm. Frederiks et H. Vreeling-sindelarova, Localization of glucose-6-phosphate dehydrogenase activity on ribosomes ofgranular endoplasmic reticulum, in peroxisomes and peripheral cytoplasm ofrat liver parenchymal cells, HISTOCHEM J, 33(6), 2001, pp. 345-353
Glucose-6-phosphate dehydrogenase activity has been localized ultrastructur
ally in fixed tissues. Activity was found in particular in association with
ribosomes of granular endoplasmatic reticulum. Biochemical studies indicat
ed that glucose-6-phosphate dehydrogenase activity is also present in the c
ytoplasm and in peroxisomes. Fixation may be held responsible for selective
inactivation of part of glucose-6-phosphate dehydrogenase activity. In the
present study, we applied the ferricyanide method for the demonstration of
glucose-6-phosphate dehydrogenase activity in unfixed cryostat sections of
rat liver in combination with the semipermeable membrane technique and in
isolated rat liver parenchymal cells. Isolated liver parenchymal cells were
permeabilized with 0.025% glutaraldehyde after NADP(+) protection of the a
ctive site of glucose-6-phosphate dehydrogenase. This treatment resulted in
only slight inactivation of glucose-6-phosphate dehydrogenase activity. Th
e composition of the incubation medium was optimized on the basis of rapid
light microscopical analysis of the formation of reddish-brown final reacti
on product in sections. With the optimized method, electron dense reaction
product was observed in cryostat sections on granular endoplasmic reticulum
, in mitochondria and at the cell border. However, the ultrastructural morp
hology was rather poor. In contrast, the morphology of incubated isolated c
ells was preserved much better. Electron dense precipitate was found on rib
osomes of the granular endoplasmic reticulum, in peroxisomes and the cytopl
asm, particularly at the periphery of cells. In conclusion, our ultrastruct
ural study clearly demonstrates that it is essential to use mildly-fixed ce
lls to allow detection of glucose-6-phosphate dehydrogenase activity in all
cellular compartments where activity is present.