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

Abstract

S U M M A R Y Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.

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“…Thiamine pyrophosphatase is a trans-Golgi marker enzyme [40,42]. Recent studies indicate that the thiamine dependent enzyme transketolase is localized around the endoplasmic reticulum (ER) [4]. The ER is involved in post-translational protein processing and transport.…”

The pentose phosphate pathway: An antioxidant defense and a crossroad in tumor cell fate

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Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29

“…Control reactions were incubated in the absence of substrate and coenzyme. 30,31 In situ determination of transketolase activity was performed according to Boren et al 32 Incubation medium contained 18% (w/ v) PVA in 50 mM Tris-HCl buffer, pH 7.6, 5 mM sodium azide, 7.5 mM NAD 1 , 3.7 mM KH 2 PO 4 , 5 mM MgCl 2 , 0.45 mM methoxyphenazine methosulphate, 100 lL substrate mixture (prepared as described above) per mL incubation medium, 0.1 mM TPP and 5 mM NBT. The medium was freshly prepared just before incubation and NBT was added after being dissolved in a heated mixture of DMF and ethanol (1:1) (final dilution of each solvent in the medium was 2% v/v).…”