An improved method for the histochemical localization of glucose-6-phoshate dehydrogenase in animal and plant tissues.

Abstract

An improved procedure is reported for the histochemical localization of glucose-6-phosphate dehydrogenase in fresh-frozen sections of animal and plant tissues. Used in this procedure are an incubation medium and an inert high-molecular-weight polyvinyl alcohol as a stabilizer to check enzyme diffusion. The incubation medium consists of 10 mg each of nicotinamide adenine dinucleotide phosphate and nitroblue tetrazolium chloride and 60 mg of glucose-6-phosphate per 20 ml of 22% polyvinyl alcohol solution in 0.2 M Tris-maleate buffer, pH 7.2. Use of this medium has given consistent results with the tissues tested. Because the use of an electron-transfer agent in the incubation medium often resulted in enzyme diffusion, its use in glucose-6-phosphate dehydrogenase localization is considered undersirable. Incubation time of 15 to 30 min for animal tissue and 20 to 45 min for plant tissue of 37degreesC was found to be optimal for producing the characteristic blue formazan at the reaction sites.

Glucose-6-phosphate dehydrogenase in rat lung alveolar epithelial cells. An ultrastructural enzyme-cytochemical study

“…Type I cells were devoid of activity. The following observations coincided well with the present findings: 1) Previous light microscopic nitroblue tetrazolium-histochemistry showed G6PD labeling in rat type II alveolar cell cytoplasm (Negi and Stephens 1977); 2) biochemical data also indicated that rat type II cells contained much higher levels of G6PD activity compared to whole lung tissue homogenates (Forman and Fisher 1981;Freeman et al, 1986); 3) the subcellular localization pattern of enzyme-cytochemically detectable G6PD in tissues other than the lung (i.e. the rat liver, the human placenta) was identical to that in the rat type II cells observed here (Ishibashi et al, 1999;Matsubara et al, 2001a;b), and; 4) biochemical (Ozols, 1993) and immuno-electron microscopic (Ninfali et al, 2000) studies demonstrated that G6PD resided both in cytoplasm and in close association with ER in rabbit hepatocytes and rat central nervous system neurons, respectively.…”

“…(Forman and Fisher 1981;Freeman et al, 1986). Alhough a previous light microscopic study also suggested the presence of G6PD in rat type II alveolar cells (Negi and Stephens 1977), definite morphological evidence for the presence of G6PD within alveolar cells has not been reported. Is cytochemically detectable G6PD present in alveolar epithelial cells?…”

Studies on Cataractogenesis in Humans and in Rats with Alloxan-Induced Diabetes

“…HK and keto-HK localization probably reflects a lack of reliable and reproducible procedures. The method used in our study differs from the recently mod ified method of Negi and Stephens [1977] in the use of aldehydes (3% glutaraldehyde and 3% formaldehyde) as fixative which preserved the tissue and helped to insolubilize the enzyme at the specific site in the tissue. The use of polyvinyl alcohol further stabilized the tissue and its soluble enzyme.…”

“…10 mg; (d) glucose-6-phosphate 60 mg. The details of the procedure arc as described by Negi and Stephens (1977). The medium without glucose-6-phosphate served as control.…”

Glucose-6-phosphate dehydrogenase activity in spinach as measured by image analysis: A new approach for plant enzyme histochemistry

“…Moreover, in SAMs of Brassica campestris, formazan deposition was decreased when PMS was added to the reaction medium for G6PDH, suggesting that PMS could act as an inhibitor (Orr, 1985). It has also been reported that PMS causes diffused formazan production in sections of plant tissues (Negi & Stephens, 1977).…”