Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

Abstract

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett's brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells.

The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas disease

“…These experiments, together with G6PDH-GFP fusion analysis, also suggested that the enzyme is probably associated to the Golgi complex in epimastigotes and metacyclic trypomastigotes (Igoillo-Esteve 2005). This localization is not completely unusual since it has been previously suggested for the G6PDH of rabbit intestine cells (Ishibashi et al 1999). Nevertheless, its biological relevance is not very clear until now; we suggest that in T. cruzi a G6PDH fraction associated with the Golgi complex would be indirectly protecting the lipid peroxidation in the organelle membranes via the microsomal Tc-GPXII (Wilkinson et al 2002b).…”

In Situ Localization of Transketolase Activity in Epithelial Cells of Different Rat Tissues and Subcellularly in Liver Parenchymal Cells

“…Biagiotti et al (2001) propose a number of functions for the high G6PD activity in these cells, which are related with the supply of NADPH for the NO synthase reaction and the production of neurosteroids (Biagiotti et al 2003). The rather low TK activity in the cortex of adrenal gland is in contrast with the very high G6PD activity in this part (Berchtold 1979;Ishibashi et al 1999), suggesting that NADPH is produced for biosynthesis of hormones rather than recycling of carbons.…”

Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry

“…Ultrathin sections were prepared using an LKB ultrotome, and observed under a Hitachi H-7000 transmission electron microscope. G6PD activity is visible as electrondense deposits of copper ferrocyanide via the following mechanism; G6P (the substrate) oxidation by enzyme G6PD generates free electron (H+), which is transferred to NADP and further to phenazine methosulfate (an exogenous electron carrier), finally trapped by ferricyanide, leading to electron-dense copper-ferrocyanide formation at the exact site of G6PD enzyme molecule (Ishibashi et al 1999). Cytochemical control experiments were performed as follows: (1) Sections were incubated in a medium (a) lacking substrate (G6P), (b) devoid of NADP, or (c) containing 2.0 mM p-chloromercuribenzoate (an inhibitor of G6PD activity); and (2) Sections were heated at 100 °C for 10 min, then incubated in the complete reaction medium.…”

“…After a wash in a cacodylate buffer (0.1 M, pH 7.4) for 3 hrs, the samples were cut into 40-µm sections with either a Vibratome or a freezing microtome. Cytochemical detection of G6PD was performed as previously described (Ishibashi et al 1999;Matsubara et al 2001a;2001b;in press). In brief, sections were incubated for 60 mins at 37°C in the dark in a reaction medium consisting of 10.5 mM G6P (disodium salt), 70 mM phosphate buffer (pH 7.2), 1.3 mM NADP, 10 mM sodium citrate, 1.5 mM copper sulfate, 0.5 mM potassium ferricyanide, 1.0 mM phenazine methosulfate, and 267 mM sucrose.…”

“…Although light microscopic enzyme-histochemistry roughly localized G6PD to Kupffer cells in rats (Hosemann et al 1979), definite morphological evidence of its presence in these cells at the electron microscopic level has not been reported. We have investigated the subcellular localization of G6PD in rat Kupffer cells, using a newly developed G6PD enzyme-cytochemistry (the copper ferrocyanide method) (Ishibashi et al 1999). …”