Electron Microscopic Demonstration of Dehydrogenase Activity With a New Osmiophilic Ditetrazolium Salt (Tc-Nbt)

Seligman, Arnold M.; Ueno, H.; Morizono, Yoshihisa; Wasserkrug, Hannah L.; Katzoff, Lionel; Hanker, Jacob S.

doi:10.1177/15.1.1

Cited by 81 publications

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“…The existence of active and nonactive mitochondria in the same cell was described by Seligman et al (8) for animal tissues and was also found in cells of normal root tips (Fig. 2).…”

Section: Discussionsupporting

confidence: 82%

The Effect of Water Stress on Mitochondria of Root Cells

1970

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Water loss from root cells of Zea mays induced by exposure to severe water stress, caused a reduction in the respiratory rate (02 consumption) and at the same time an increase in activity of the mitochoDdrial cytochrome oxidase (histochemical oxidation of diaminobenzidine). The connections between these changes and the changes in the fine structure of the mitochondria occuring in the stressed roots are discussed.Porter-Blum ultramicrotome and examined with an RCA EMU-3G electron microscope.For isolation of mitochondria, 150 root tips (5 mm long) were ground in a prechilled mortar with 3.0 ml of phosphate buffer (0.05 M, pH 7.5) containing 0.4 M sucrose and 3 x 10-' M MgSO4. The homogenate was centrifuged for 5 min at 2,000 g; the mitochondria were sedimented from the supernatant by centrifugation at 20,000g for 30 min and resuspended in 1.0 ml of the isolation medium. The cytochrome oxidase activity was determined by measuring spectrophotometrically the oxidation of reduced cytochrome c as described by Smith (10). The results are given as specific activity which was obtained by relating the calculated first order reaction constant to milligrams of protein. Oxygen uptake was measured in a Warburg respirometer.In previous reports (5, 6) the effect of dehydration on mitochondrial fine structure was described. A change in shape, a decrease in matrix density, and a decrease in the visibility of the cristae were reported.In order to correlate these structural changes with biochemical function, experiments were carried out in which cytochrome oxidase activity was tested in vitro, in isolated mitochondria, using biochemical methods and in situ by cytochemical methods. In addition, the rate of 02 uptake by entire root tips was investigated. MATERIALS AND METHODSSeeds of Zea mays (var. Neve Yaar 22) were germinated on wet filter paper for 15 hr in the dark at 250. Root tips, 5 mm long, were cut off the main rootlets and served as the experimental material.Groups of such root tips were weighed and exposed to water stress above concentrated solutions of NaCl as previously described (6). Water loss was determined by weighing and expressed as a percentage of initial weight.The cytochrome oxidase activity in the cell was localized in prefixed root tips by reaction with diaminobenzidine, as described by Seligman et al. (7). Root tips 2 mm long were prefixed for 60 min in 4% formaldehyde at 20. The formaldehyde was prepared by depolimerization of p-formaldehyde (1).Sections 100 thick were cut from the fixed root tips with a freezing microtome. The sections were incubated for 30 min at 300 with a reaction mixture containing 10 ml of phosphate buffer (0.05 M, pH 7.5), 0.5 mg of diaminobenzidine, 10 mg of cytochrome c, and 50 mg of sucrose. Then the sections were washed for 15 min with the phosphate buffer containing 5% sucrose and exposed for 60 min at 350 to 2% OS04 (dissolved in phosphate buffer). The sections were dehydrated in graded ethanol and embedded in Epon (4). Thin sections were cut with a 1 This work is a part ...

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“…The existence of active and nonactive mitochondria in the same cell was described by Seligman et al (8) for animal tissues and was also found in cells of normal root tips (Fig. 2).…”

Section: Discussionsupporting

confidence: 82%

The Effect of Water Stress on Mitochondria of Root Cells

1970

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“…It was of interest to determine whether succinic dehydrogenase, which ordinarily donates electrons via cytochrome b to nitro-containing tetrazolium salts (14), had a similar or opposite localization for its electron-donating activity as compared to cytochrome oxidase. However, doubt has been cast on our earlier interpretations of the observation of dense, contracted mitochondria in dehydrogenase preparations of fresh rat heart made with an osmiophilicditetrazolium salt (TC-NBT) (21). 4 Haydon 4 Material on the market being sold as TC-NBT by Nutritional Biochemicals Corporation, Cleveland, O., is not osmiophilic and melts 30 0 C lower than our material.…”

Section: Discussionmentioning

confidence: 78%

Nondroplet Ultrastructural Demonstration of Cytochrome Oxidase Activity With a Polymerizing Osmiophilic Reagent, Diaminobenzidine (Dab)

Seligman

Karnovsky

Wasserkrug

et al. 1968

The Journal of Cell Biology

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855

290

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A new method for demonstrating cytochrome oxidase activity, based upon the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic reaction product, has improved the localization of this enzyme over methods based upon the Nadi reaction, in both the light and electron microscopes. The reaction product occurs in nondroplet form, which more accurately delineates the localization of cytochrome oxidase in mitochondria of heart, liver, and kidney. In electron microscopic preparations the excess reaction product is found to overflow into the intracristate spaces and into the outer compartment between inner and outer limiting mitochondrial membranes. This finding suggests that the enzymatic activity of cytochrome c is located on the inner surface of the intracristate space which is the outer surface of the inner mitochondrial membrane. Succinic dehydrogenase activity has also been located at this site by using an osmiophilic ditetrazolium salt, TC-NBT. Considered together, the sites of reactivity of both parts of the respiratory chain have implications for the chemiosomotic hypothesis of Mitchell who suggests a mechanism of energy conservation during electron transport in the respiratory chain of the mitochondrion.

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“…Unstained sections were also studied. This procedure has also been used to reveal underlying ultrastructure for other enzymatic methods (5,21,22).…”

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confidence: 99%

Ultrastructural Demonstration of Cytochrome Oxidase Activity by the Nadi Reaction With Osmiophilic Reagents

Seligman

Plapinger

Wasserkrug

et al. 1967

The Journal of Cell Biology

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A new method for the subcellular and cytochemical demonstration of cytochrome oxidase has been developed with the introduction of N-benzyl-p-phenylenediamine (BPDA) and the discovery that indoanilines are osmiophilic. These indoanilines produced upon oxidation of BPDA in the presence of naphthols are highly colored compounds that yield electronopaque coordination polymers of osmium (osmium black) that are amorphous, insoluble in water, and in organic solvents. The best methods for preparing rat tissue were in decreasing order: fixation in formaldehyde solution, fresh tissue slices, and frozen sections of fresh or fixed tissue. Ultrathin sections were counterstained by bridging with the thiocarbohydrazide-osmium tetroxide (T-O) procedure for enhancing underlying membranous structures. Cytochrome oxidase activity was noted primarily in mitochondria and occasionally in sarcotubules of heart, in mitochondria and occasionally in infoldings of the plasma membrane of renal tubular cells, and in mitochondria and, to a great extent, in endoplasmic reticulum of hepatic cells. Cytochrome oxidase activity produced deposits in droplet form, whereas dehydrogenase activity resulted in uniform staining of mitochondrial cristae, as recently demonstrated with an osmiophilic tetrazolium salt. Even more recently we have succeeded in demonstrating cytochrome oxidase activity in nondroplet staining on mitochondrial cristae with an osmiophilic benzidine-type reagent that apparently polymerizes upon oxidation (to be published later).In order to develop new cytochemical methods for electron microscopy that would supplement the metal salt methods for hydrolytic enzymes and possibly avoid some of their pitfalls, we have introduced new histochemical principles (5,7,8,20). One principle is based upon the use of organic reactions that yield insoluble pigments which possess an osmiophilic group. The immediate conversion of the reaction product into osmium black on exposure to osmium tetroxide helps to diminish the artifacts due to possible diffusion of the organic reaction product or due to special affinities of reaction product such as lipid solubility. The osmium black so produced is a coordination polymer of osmium (6) and has ideal properties for both light and electron microscopy.In order to do the same for cytochrome oxidase, we could have introduced the osmiophilic group into either of the two components of the Nadi reaction. In our earlier experiments (7,8,20),

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Electron Microscopic Demonstration of Dehydrogenase Activity With a New Osmiophilic Ditetrazolium Salt (Tc-Nbt)

Cited by 81 publications

References 0 publications

The Effect of Water Stress on Mitochondria of Root Cells

The Effect of Water Stress on Mitochondria of Root Cells

Nondroplet Ultrastructural Demonstration of Cytochrome Oxidase Activity With a Polymerizing Osmiophilic Reagent, Diaminobenzidine (Dab)

Ultrastructural Demonstration of Cytochrome Oxidase Activity by the Nadi Reaction With Osmiophilic Reagents

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