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.

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. 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 characterization of neuronal NADPH-diaphorase.

1989 ◽  
Vol 37 (5) ◽  
pp. 653-661 ◽  
Author(s):  
B T Hope ◽  
S R Vincent
Keyword(s):  
Nadph Diaphorase ◽  
Rat Striatum ◽  
Tetrazolium Salt ◽  
Diaphorase Activity ◽  
Central Neurons ◽  
Membrane Bound ◽  
Pre Treatment ◽  
Dt Diaphorase ◽  
Discrete Populations

We examined the properties of neuronal NADPH-diaphorase in sections of rat striatum, using histochemical procedures. NADPH-diaphorase histochemistry stained discrete populations of central neurons and provided a Golgi-like image of the neurons exhibiting this activity. The NADPH-diaphorase reaction appeared to be enzyme catalyzed, since it was abolished by pre-treatment with proteases, heat, and acid or alkaline denaturation. Under anaerobic conditions, any tetrazolium salt with a redox potential more positive than NADPH could be reduced by the enzyme. NADPH-diaphorase activity was sensitive to inhibition by sulfhydryl reagents but was unaffected by metal chelators, superoxide dismutase, and catalase. Therefore, the enzyme is unlikely to be a metalloenzyme or to reduce tetrazoliums by producing superoxide anions or hydrogen peroxide. Various analogues of beta-NADPH could be used by the enzyme; however, beta-NADH, which can be used by DT-diaphorase, was ineffective. The enzyme was also resistant to dicumarol, an inhibitor of DT-diaphorase activity. Electron microscopy indicated that the NADPH-diaphorase reaction resulted in staining of various membranous organelles. We conclude that neuronal NADPH-diaphorase is a membrane-bound enzyme distinct from DT-diaphorase and other known enzymes with diaphorase activity. The histochemical characteristics presented here should now enable meaningful biochemical studies of neuronal NADPH-diaphorase to be undertaken.

scholarly journals The Histochemical Localization of Triphosphopyridine Nucleotide Diaphorase

1958 ◽  
Vol 4 (4) ◽  
pp. 467-474 ◽  
Author(s):  
Marvin M. Nachlas ◽  
Donald G. Walker ◽  
Arnold M. Seligman
Keyword(s):  
Macula Densa ◽  
Histochemical Method ◽  
Histochemical Localization ◽  
Kidney Cortex ◽  
Tetrazolium Salt ◽  
Tissue Sections ◽  
Isocitric Dehydrogenase ◽  
Diaphorase Activity

A histochemical method is described for the localization of triphosphopyridine nucleotide diaphorase using a recently synthesized tetrazolium salt (Nitro-BT). By virtue of the favorable histochemical properties of this reagent, it has been possible to demonstrate that whereas DPN diaphorase is usually restricted to the mitochondria, the TPN diaphorase activity of corresponding cells was distributed throughout the cytoplasm in granules too fine to be considered mitochondria. Furthermore, although the diaphorase alone is responsible for the passage of electrons from TPNH to the tetrazole, it has been found that sites of activity of different TPN-linked dehydrogenases can be visualized in tissue sections, and characteristic loci for each enzyme may be observed. For example, whereas TPN diaphorase and isocitric dehydrogenase have an extensive distribution in the kidney cortex, 6-phosphogluconic dehydrogenase is limited to the cells of the macula densa.

scholarly journals CYTOCHEMICAL LOCALIZATION OF TWO GLYCOLYTIC DEHYDROGENASES IN WHITE SKELETAL MUSCLE

1966 ◽  
Vol 29 (1) ◽  
pp. 113-128 ◽  
Author(s):  
H. Dariush Fahimi ◽  
Morris J. Karnovsky
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Electron Flow ◽  
Aqueous Media ◽  
Cytochemical Localization ◽  
Diaphorase Activity ◽  
Adductor Magnus ◽  
Glycolytic Activity ◽  
Final Electron ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Soluble Enzymes

The cytochemical localization, by conventional methods, of lactate and glyceraldehyde-3-phosphate dehydrogenases is limited, firstly, by the solubility of these enzymes in aqueous media and, secondly, by the dependence of the final electron flow from reduced nicotinamide-adenine dinucleotide (NADH) to the tetrazolium on tissue diaphorase activity: localization is therefore that of the diaphorase, which in rabbit adductor magnus is mitochondrial. NADH has been found to have great affinity to bind in the sarcoplasmic reticulum, and, therefore, if it is generated freely in the incubation media containing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy-4,4'-phenylene)-ditetrazolium chloride (TNBT) and N-methyl phenazonium methyl sulfate (PMS), it can bind there and cause a false staining. Since such a production of NADH can readily occur in the incubation media for glycolytic dehydrogenases due to diffusion of these soluble enzymes from tissue sections, the prevention of enzyme solubilization is extremely important. Fixation in formaldehyde prevented such enzyme diffusion, while at the same time sufficient activity persisted to allow for adequate staining. The incubation media contained PMS, so that the staining system was largely independent of tissue diaphorase activity. Application of these methods to adductor magnus of rabbit revealed by light microscopy, for both enzymes, a fine network which was shown by electron microscopy to represent staining of the sarcoplasmic reticulum. Mitochondria also reacted. These findings add further support for the notion that the sarcoplasmic reticulum is probably involved in glycolytic activity.

scholarly journals Cytochemical localization of acid phosphatase and trimetaphosphatase activities in exocrine acinar cells.

1980 ◽  
Vol 28 (1) ◽  
pp. 78-81 ◽  
Author(s):  
C Oliver
Keyword(s):  
Acid Phosphatase ◽  
Lysosomal Enzyme ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Metal Chelation ◽  
Acid Hydrolases ◽  
Cytochemical Localization ◽  
Capture Method ◽  
Secondary Lysosomes

Acid phosphatase activity, a lysosomal marker, is commonly demonstrated using the Gomori technique with cytidine 5'-monophosphate or beta-glycerophosphate as substrate. Using this lead capture method on mouse and rat exorbital lacrimal, parotid, and pancreatic acinar cells, reaction product was localized in GERL, forming secretory granules, and secondary lysosomes. However, a different cytochemical localization was observed for inorganic trimetaphosphatase, another lysosomal enzyme. When the technique for trimetaphosphatase activity, a metal chelation method, was applied to exocrine acinar cells, reaction produce was conspicuously absent from GERL and forming secretory granules, but was present in secondary lysosomes, occasionally in Golgi saccules, and in previously unreported basal elongated lysosomes. The differences in the localization of the two enzymatic activities emphasizes the importance of employing more than one substrate where possible, and raises questions concerning the mechanism of delivery of acid hydrolases to secondary lysosomes.

scholarly journals The cytochemical localization of succinic dehydrogenase in the sperm mitochondria of the gastropod Biomphalaria glabrata. A comparison of two methods.

1975 ◽  
Vol 23 (1) ◽  
pp. 75-79 ◽  
Author(s):  
B J Bogitsh
Keyword(s):  
Electron Transport ◽  
Nicotinamide Adenine Dinucleotide ◽  
Succinic Dehydrogenase ◽  
Biomphalaria Glabrata ◽  
The Other ◽  
Tetrazolium Salt ◽  
Cytochemical Localization ◽  
Mitochondrial Derivative ◽  
The Matrix ◽  
Transport Inhibitors

The mitochondrial derivative of the sperm of the gastropod pulmonate Biomphalaria glabrata was studies to ascertain succinic dehydrogenase localization cytochemically. Two techniques were compared. One technique depends on a tetrazolium salt that yields an osmiophilic formazan upon reduction. The other technique is dependent on the reduction of copper ferricyanide. The effects of several electron transport inhibitors were studied. The reaction product observed in the matrix of the mitochondrial derivative using the former technique is sensitive to rotenone and is believed to be nicotinamide adenine dinucleotide-dependent. The reaction product observed in the intracristal spaces using the copper ferricyanide method is insensitive to rotenone and is believed to cytochemically demonstrate succinic dehydrogenase in this material.

scholarly journals Cytochemical localization of certain oxidative enzymes

1962 ◽  
Vol 110 (1) ◽  
pp. 19-27 ◽  
Author(s):  
James L. Conklin ◽  
Maynard M. Dewey ◽  
Raymond H. Kahn
Keyword(s):  
Oxidative Enzymes ◽  
Cytochemical Localization

The Ultrastructural Localization of Acid and Alkaline Phosphatase Via Sulfhydryl Reduction of a Nonosmiophilic Tetrazolium Salt to an Osmiophilic Formazan

1973 ◽  
Vol 31 ◽  
pp. 694-695
Author(s):  
W. Allen Shannon ◽  
Yoshinobu Hoshino ◽  
Arnold M. Seligman
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Metal Salt ◽  
Ultrastructural Localization ◽  
Enzymatic Activities ◽  
Tetrazolium Salt ◽  
Lysosomal Hydrolases ◽  
Cytochemical Localization ◽  
New Approach ◽  
Acid And Alkaline Phosphatase

The metal salt capture methods for acid phosphatase and alkaline phosphatase with numerous variations based principally on the Gomori method have been useful methods for direct cytochemical visualization of the enzymes. However, the possibilities of nonenzymatic deposition of reaction product, possible inhibition of enzymatic activities, and other suggested weaknesses of the methods which are found in the literature give reason to develop a new approach to the cytochemical localization of these enzymes. Other attempts along these lines have thus far been more or less partially successful. More recently Hanker et al. have demonstrated lysosomal hydrolases by the formation of osmium blacks based on synthetic substrates which initially deposit Hatchett's brown (cupric ferrocyanide) at the reactive sites. The synthetic substrate', 2-naph-thylthiolphosphate (NTP), developed by Seligman et al. to use in the demonstration of acid and alkaline phosphatase and subsequently used by Hanker et al.

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