Localization of glucose-6-phosphate dehydrogenase activity on ribosomes of granular endoplasmic reticulum, in peroxisomes and peripheral cytoplasm of rat liver parenchymal cells.
Glucose-6-phosphate dehydrogenase activity has been localized ultrastructurally in fixed tissues. Activity was found in particular in association with ribosomes of granular endoplasmatic reticulum. Biochemical studies indicated that glucose-6-phosphate dehydrogenase activity is also present in the cytoplasm 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 active site of glucose-6-phosphate dehydrogenase. This treatment resulted in only slight inactivation of glucose-6-phosphate dehydrogenase activity. The composition of the incubation medium was optimized on the basis of rapid light microscopical analysis of the formation of reddish-brown final reaction 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 morphology was rather poor. In contrast, the morphology of incubated isolated cells was preserved much better. Electron dense precipitate was found on ribosomes of the granular endoplasmic reticulum, in peroxisomes and the cytoplasm, particularly at the periphery of cells. In conclusion, our ultrastructural study clearly demonstrates that it is essential to use mildly-fixed cells to allow detection of glucose-6-phosphate dehydrogenase activity in all cellular compartments where activity is present.
Frederiks
W M
WM
Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, The Netherlands.
Vreeling-Sindelárová
H
H
D000818:Animals; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology; D004577:Electron Probe Microanalysis; D018870:Endoplasmic Reticulum, Rough / Q000201:enzymology* / Q000648:ultrastructure; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism* / Q000648:ultrastructure; D022781:Hepatocytes / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D020675:Peroxisomes / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D012270:Ribosomes / Q000201:enzymology* / Q000648:ultrastructure
D005954:Glucosephosphate Dehydrogenase