scholarly journals The Cytochemical Localization of Oxidative Enzymes

1958 ◽  
Vol 4 (6) ◽  
pp. 753-760 ◽  
Author(s):  
R. Hess ◽  
D. G. Scarpelli ◽  
A. G. E. Pearse
Keyword(s):  
Oxidative Enzymes ◽  
Tetrazolium Salt ◽  
Differential Centrifugation ◽  
Mitochondrial Localization ◽  
Cytochemical Localization ◽  
Tissue Sections ◽  
Final Electron ◽  
High Concentrations ◽  
Distinct Distribution ◽  
Final Electron Acceptor

Methods are presented for the intramitochondrial localization of various diphosphopyridine nucleotide and triphosphopyridine nucleotide-linked dehydrogenases in tissue sections. The cytochemical reactions studied involve the oxidation of the substrates by a specific pyridino-protein. The electron transfer of tetrazolium salt is mediated by the diaphorase system associated with the dehydrogenase. The final electron acceptor was either p-nitrophenyl substituted ditetrazole (nitro-BT) or N-thiazol-2-yl monotetrazole (MTT), the latter giving rise to metal formazan in the presence of cobaltous ions. Mitochondrial localization of the formazan precipitate could be achieved by using hypertonic incubating media containing high concentrations of substrate and co-enzyme. A fast reduction of tetrazolium salt was obtained by chemically blocking the respiratory chain enzymes beyond the flavoproteins. Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates. The present findings in tissue sections are discussed in conjunction with existing biochemical evidence from differential centrifugation experiments.

scholarly journals HISTOCHEMICAL DEMONSTRATION OF 3α-HYDROXYSTEROID DEHYDROGENASE ACTIVITY

1966 ◽  
Vol 14 (1) ◽  
pp. 77-83 ◽  
Author(s):  
KÁROLY BALOGH
Keyword(s):  
Leydig Cells ◽  
Hydroxysteroid Dehydrogenase ◽  
Histochemical Demonstration ◽  
Female Rats ◽  
Ventral Prostate ◽  
Tetrazolium Salt ◽  
Rat Tissues ◽  
Final Electron ◽  
Nucleotide Specificity ◽  
Final Electron Acceptor

The reversible oxidation of 3α-hydroxysteroids to their corresponding 3-keto forms comprises an important step in the metabolism of C19-steroids. The described techniquue demonstrates the activity of the enzyme catalyzing this reaction, with the use of androsterone as a substrate and a tetrazolium salt as the final electron acceptor. The enzyme is specific for 3α-hydroxysteroids; there was no histochemical reaction with epiandrosterone, the β isomer of androsterone. Since 3α-hydroxysteroid dehydrogenase is soluble in aqueous solutions, it was necessary to increase the osmolarity of the incubation medium by adding polyvinylpyrrolidone in a final concentration of 20%. Although the enzyme has a dual nucleotide specificity, no appreciable differences were seen in its distribution pattern in rat tissues with either NAD or NADP as a coenzyme. In adult female rats, enzyme activity was present in the liver, kidneys and clitoral glands. In mature males, diformazan deposits were observed in the liver, kidneys, preputiai glands, epididymis, ventral prostate and Leydig cells.

scholarly journals THE CYTOCHEMICAL LOCALIZATION OF GLYCOLYTIC AND OXIDATIVE ENZYMES WITHIN MITOCHONDRIA OF SPERMATOZOA OF SOME PULMONATE GASTROPODS

1970 ◽  
Vol 18 (11) ◽  
pp. 783-793 ◽  
Author(s):  
WINSTON A. ANDERSON ◽  
PAUL PERSONNE
Keyword(s):  
Reductase Activity ◽  
Periodic Acid ◽  
Oxidative Enzymes ◽  
Tetrazolium Salt ◽  
Phenazine Methosulfate ◽  
Electron Microscopic ◽  
Cytochemical Localization ◽  
Cytochemical Study ◽  
Mitochondrial Derivative ◽  
The Matrix

In this electron microscopic cytochemical study, the periodic acid-thiosemicarbazide-silver proteinate procedure was used to demonstrate glycogen stores within the mitochondrial derivative of sperm of pulmonate gastropods. In the presence of phenazine methosulfate and tetrazolium salt, enzymatic activity for glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase is shown in the matrix and in the compartment containing glycogen, but in the absence of phenazine methosulfate, tetrazolium reductase activity in the matrix is emphasized. Activity for NADH2-tetrazolium reductase and succinate dehydrogenase is also demonstrated in the matrix. Using 3,3'-diaminobenzidine tetra-HCl, cytochrome c oxidase activity is shown in the paracrystalline mitochondrial structure. The interrelation between glycolytic and oxidative pathways in this highly compartmentalized mitochondrion is considered.

scholarly journals The Cytochemical Localization of Oxidative Enzymes

1958 ◽  
Vol 4 (6) ◽  
pp. 747-752 ◽  
Author(s):  
D. G. Scarpelli ◽  
R. Hess ◽  
A. G. E. Pearse
Keyword(s):  
Cellular Localization ◽  
Oxidative Enzymes ◽  
Tetrazolium Salt ◽  
Direct Transfer ◽  
Metal Chelation ◽  
Cytochemical Localization ◽  
Black Metal ◽  
Respiratory Inhibitors ◽  
Diaphorase Activity ◽  
Tetrazolium Reduction

Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 µ in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.

Peroxidase Localization in Hartmanella Trophozoites

1971 ◽  
Vol 29 ◽  
pp. 346-347 ◽  
Author(s):  
George E. Childs ◽  
Joseph H. Miller
Keyword(s):  
Hydrogen Peroxide ◽  
Ultrastructural Localization ◽  
Pathogenic Strain ◽  
Oxidative Enzymes ◽  
Differential Centrifugation ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
The Subject ◽  
Axenic Cultures

Biochemical and differential centrifugation studies have demonstrated that the oxidative enzymes of Acanthamoeba sp. are localized in mitochondria and peroxisomes (microbodies). Although hartmanellid amoebae have been the subject of several electron microscopic studies, peroxisomes have not been described from these organisms or other protozoa. Cytochemical tests employing diaminobenzidine-tetra HCl (DAB) and hydrogen peroxide were used for the ultrastructural localization of peroxidases of trophozoites of Hartmanella sp. (A-l, Culbertson), a pathogenic strain grown in axenic cultures of trypticase soy broth.

scholarly journals MITOCHONDRIAL LOCALIZATION OF OXIDATIVE ENZYMES: STAINING RESULTS WITH TWO TETRAZOLIUM SALTS

1961 ◽  
Vol 9 (1) ◽  
pp. 47-61 ◽  
Author(s):  
Alex B. Novikoff ◽  
Woo-Yung Shin ◽  
Joan Drucker
Keyword(s):  
Rat Kidney ◽  
Intracellular Localization ◽  
Oxidative Enzymes ◽  
Mitochondrial Morphology ◽  
Mitochondrial Localization ◽  
Enzyme Localization ◽  
Aqueous Interfaces ◽  
Physicochemical Factors ◽  
Lipofuscin Granules ◽  
In Cells

A comparison is made of the staining results obtained with Nitro-BT and MTT-Co++ as acceptors when DPNH is the substrate in frozen sections of cold formol-calcium-fixed rat kidney (normal and following ligation of the blood vessels) and human liver containing lipofuscin granules. The kidney results are evaluated in terms of mitochondrial morphology seen after classical mitochondrial stains and in electron micrographs. It is concluded that, except for formazan deposition on lipid-aqueous interfaces, Nitro-BT staining indicates the intracellular localization of oxidative enzymes, at least at the level of light microscopy. In contrast, the use of MTT-Co++ is not reliable for such intracellular localizations. The deposition of the formazan of MTT-Co++ is determined in large part by physicochemical factors other than enzyme localization. Despite marked abnormalities of the mitochondria in cells of the ligated kidney, MTT-Co++ formazan is generally deposited in the same dotlike fashion as in cells of normal kidney.

Reduction of selenate and selenite to elemental selenium by Wolinella succinogenes

1992 ◽  
Vol 38 (12) ◽  
pp. 1328-1333 ◽  
Author(s):  
Francisco A. Tomei ◽  
Larry L. Barton ◽  
Cheryl L. Lemanski ◽  
Thomas G. Zocco
Keyword(s):  
Stationary Growth Phase ◽  
Elemental Selenium ◽  
Bacterial Cells ◽  
Wolinella Succinogenes ◽  
Stationary Growth ◽  
X Ray ◽  
Dense Granules ◽  
Transmission Electron ◽  
Final Electron ◽  
Final Electron Acceptor

Cultures of Wolinella succinogenes were adapted to grow in the presence of 1 mM [Formula: see text] or 10 mM [Formula: see text]. Both selenium salts were reduced to red, amorphous, elemental selenium but only after the culture reached the stationary growth phase. Bacterial cells taken from a culture actively reducing selenium were examined by transmission electron microscopy and were found to have large, electron-dense granules in the cytoplasm. These granules were verified by energy-dispersive X-ray spectroscopy to consist of selenium. Wolinella succinogenes was unable to grow with [Formula: see text] or [Formula: see text] as the final electron acceptor. Key words: Wolinella, selenium, cytology, selenate.

scholarly journals CYTOCHEMICAL LOCALIZATION OF LACTIC DEHYDROGENASE IN WHITE SKELETAL MUSCLE

1964 ◽  
Vol 22 (1) ◽  
pp. 29-48 ◽  
Author(s):  
H. Dariush Fahimi ◽  
Chandra Raj Amarasingham
Keyword(s):  
Skeletal Muscle ◽  
White Muscle ◽  
Lactic Dehydrogenase ◽  
Gelatin Film ◽  
Histochemical Reaction ◽  
Cytochemical Localization ◽  
Tissue Sections ◽  
Modified Method ◽  
Muscle Extract ◽  
Adductor Magnus Muscle

The limitations of the conventional histochemical methods for localization of lactic dehydrogenase (LDH) in white skeletal muscle have been analyzed quantitatively. It is demonstrated that more than 80 per cent of LDH diffuses into the incubation medium within the first 10 minutes of incubation. Furthermore, it is confirmed that the addition of phenazine methosulfate (PMS) to the ingredients of the histochemical reaction for LDH increases substantially the capacity of the white muscle extract to reduce Nitro-BT. Based on these observations, a modified method of cytochemical localization of LDH has been developed. This method prevents the leakage of LDH from tissue sections by the application of all the ingredients of the histochemical reaction to tissue sections in a thin gelatin film. The incubation mixture contains PMS so that the staining system is independent of tissue diaphorase. The application of this method to the adductor magnus muscle of the rabbit revealed a fine reticulum in the sarcoplasm of all muscle fibers, in addition to the staining of mitochondria. The distribution of the staining suggests that LDH is localized in the sarcoplasmic reticulum.

Biochemical and regulatory properties of a respiratory island encoded by a conjugative plasmid in the extreme thermophile Thermus thermophilus

2006 ◽  
Vol 34 (1) ◽  
pp. 97-100 ◽  
Author(s):  
F. Cava ◽  
J. Berenguer
Keyword(s):  
Nadh Dehydrogenase ◽  
Current Knowledge ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Conjugative Plasmid ◽  
Nitrate Respiration ◽  
Final Electron ◽  
New Type ◽  
Regulatory Properties ◽  
Final Electron Acceptor

In the present paper, we summarize the current knowledge on the first step of the denitrification pathway in the ancestral extreme thermophilic bacterium Thermus thermophilus. In this organism, nitrate respiration is performed by a mobilizable respiratory island that encodes a new type of respiratory NADH dehydrogenase as electron donor, a tetrameric membrane nitrate reductase as final electron acceptor, two nitrate/nitrite transporters and the transcription factors required for their expression in response to nitrate and anoxia.

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