scholarly journals NONOSMIOPHILIC TETRAZOLIUM SALTS THAT YIELD OSMIOPHILIC, LIPOPHOBIC FORMAZANS FOR ULTRASTRUCTURAL LOCALIZATION OF DEHYDROGENASE ACTIVITY

1972 ◽  
Vol 20 (9) ◽  
pp. 685-695 ◽  
Author(s):  
MOSHE KALINA ◽  
ROBERT E. PLAPINGER ◽  
YOSHINOBU HOSHINO ◽  
ARNOLD M. SELIGMAN
Keyword(s):  
Dehydrogenase Activity ◽  
Osmium Tetroxide ◽  
Succinic Dehydrogenase ◽  
Ultrastructural Localization ◽  
Nitroblue Tetrazolium ◽  
Tetrazolium Salt ◽  
Succinic Dehydrogenase Activity ◽  
Tetrazolium Chloride ◽  
Tetrazolium Salts ◽  
Cytochemical Demonstration

Monotetrazolium salts were designed and prepared which incorporate a phthalhydrazide moiety to make them lipophobic and a benzothiazole moiety to make them react with osmium tetroxide after they are reduced to the corresponding formazans. The tetrazolium salts themselves do not react with osmium tetroxide under the conditions used for the cytochemical demonstration of enzyme activities. Although all of the new formazans, when dissolved in dimethylformamide, were reoxidized to the tetrazolium salts by osmium tetroxide, they were not reoxidized by osmium tetroxide when they were dissolved in tetrahydrofuran or precipitated by reduction in tissue, but gave dark complexes. One of the tetrazolium salts, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT), was readily reduced by succinic dehydrogenase activity, gave a formazan which produced a dark osmium complex relatively rapidly and gave good localization of succinic dehydrogenase activity on the membranes of mitochondria of rat myocardial cells. Although this tetrazolium salt (BSPT) was not photoreduced by fresh cells of Elodea to stain chloroplasts as seen with nitroblue tetrazolium or distyryl nitroblue tetrazolium, the 5-p-nitrophenyl derivative of BSPT was photoreduced by chloroplasts. The preparation of BSPT and its use in demonstrating succinic dehydrogenase activity ultracytochemically is given here. The preparation and ultracytochemical use of the 5-p-nitrophenyl derivative in chloroplasts will be published later.

scholarly journals The ultracytochemical demonstration of a formaldehyde-resistant dehydrogenase of leuco nitroxyl analogues.

1975 ◽  
Vol 23 (11) ◽  
pp. 840-854 ◽  
Author(s):  
W A Shannon ◽  
H L Wasserkrug ◽  
R E Plapinger ◽  
D Lynm ◽  
A M Seligman
Keyword(s):  
Skeletal Muscle ◽  
Electron Microscope ◽  
Dehydrogenase Activity ◽  
Electron Density ◽  
Osmium Tetroxide ◽  
Tetrazolium Salt ◽  
Hydrogen Ions ◽  
Tetrazolium Chloride

A dehydrogenase which is relatively stable in formaldehyde fixative is demonstrated ultracytochemically by the reduction of various leuco nitroxyl analogues in rat hepatic, renal, myocardial, skeletal muscle and prostatic tubuloalveolar glandular tissues. The nonosmiophilic tetrazolium salt, t-(2'-benzothiazolyl)-5-styryl-3-(4'-phtalhydrazidyl) tetrazolium chloride, is subsequently reduced to an insoluble osmiophilic formazan by the hydrogen ions resulting from the dehydrogenase activity. Exposure of the formazan to osmium tetroxide results in electron density enabling visualization of the reaction product in the electron microscope. Known inhibitors of various dehydrogenases were utilized in an attempt to determine the existence and/or extent of any specific characteristics of the dehydrogenase(s) involved.

Mitochondrial populations and succinic dehydrogenase in the heart of a hibernator

1968 ◽  
Vol 46 (6) ◽  
pp. 911-913 ◽  
Author(s):  
Marilyn L. Zimny ◽  
James E. Moreland
Keyword(s):  
Electron Microscopy ◽  
Body Temperature ◽  
Dehydrogenase Activity ◽  
Electron Micrographs ◽  
Osmium Tetroxide ◽  
Succinic Dehydrogenase ◽  
Ground Squirrels ◽  
Succinic Dehydrogenase Activity ◽  
Control Value ◽  
Experimental Group

Thirteen-lined ground squirrels, Citellus tridecemlineatus, hibernating at 3–5 °C, awakened at 25–27 °C. Ten animals were killed for each experimental group as follows: (1) [Formula: see text] awakening to a body temperature of 9–13 °C, (2) 15 min awakening to 13–15 °C, and (3) 30 min awakening to 16–19 °C. At death a sample of the heart's apex was fixed in osmium tetroxide, dehydrated, and embedded in Vestopal-W for electron microscopy, and a 10% w/v water homogenate was made of the rest of the organ. Mitochondrial counts and measurements of dimensions were made on electron micrographs of at least 10 fields of each sample, and duplicate samples of homogenate were incubated at 37 °C and assayed spectrophotometrically for succinic dehydrogenase activity. When compared with data on control animals, during awakening the number of mitochondria per field decreased 25% (P < 0.01). Mitochondrial length increased 10% (P < 0.05) and width increased 28% (P < 0.01). In tissue obtained from hibernating animals, the succinic dehydrogenase activity was 60% of the control. This value decreased further to 42% of the control value during the first 7.5 min of awakening, thereafter returning to 94% of the control by 15 min.

The Localization of Dehydrogenase Activity and Sulphydryl Groups in Wool and Hair Follicles by the use of Tetrazolium Salts

1953 ◽  
Vol s3-94 (27) ◽  
pp. 253-268
Author(s):  
G. E. ROGERS
Keyword(s):  
Dehydrogenase Activity ◽  
Metabolic Activity ◽  
Succinic Dehydrogenase ◽  
Hair Follicles ◽  
Frozen Sections ◽  
Tetrazolium Chloride ◽  
Tetrazolium Salts ◽  
Hair Matrix ◽  
Sulphydryl Groups ◽  
Wool Follicle

Three tetrazolium salts are used as histochemical indicators of dehydrogenase activity in the skin of sheep and other mammals. Of these compounds 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride (INT) is shown to be the most satisfactory. It enables various histological structures of the wool follicle to be conveniently demonstrated. In the presence of appropriate substrates and DPN and ATP, the intracellular deposition of the granular formazan in frozen sections has revealed the distribution of succinic, α-glycerophosphoric, lactic, and malic dehydrogenases, and regions where some phosphorylated carbohydrate intermediates promote reduction. Metabolic activity in the Malpighian layers and in the undifferentiated epidermal cells of the hair matrix reduces INT enzymatically up to the pre-keratinous zone. Here the protein-bound sulphydryl groups reduce INT and produce intense red staining which is easily distinguished from the granular enzymic staining. The distribution of cytochrome oxidase in the wool follicle resembles the distribution of dehydrogenase activity. INT is used for the estimation of succinic dehydrogenase in skin and kidney tissues.

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