Demonstration Of Cytochrome Oxidase Activity Research Articles

Rapid cytochemical demonstration of cytochrome oxidase activity in pathogenic bacteria.

To develop and assess a cytochemical technique for the light microscopical detection of oxidase activity in pathogenic bacteria. Live bacterial cells were deposited on to aminopropylsilane treated glass coverslips by centrifugation, dried, then reacted with either 1% (w:v) n,n,n',n'-tetramethyl-p-phenylene diamine (TPD) or 5 mM diaminobenzidine (DAB) at 37 degrees C. The preparations were mounted in 50% glycerol and assessed by brightfield microscopy. An optimised DAB procedure (5 minutes of drying at 37 degrees C and 10 minutes of reaction time) was applied to 44 strains of organisms commonly associated with infections in man and to two fresh positive blood culture containing Gram negative bacilli. TPD gave no discernible localised reaction product and was not investigated further. With DAB, oxidase positive cells (brown) were clearly differentiated from oxidase negative cells (colourless) even in mixed preparations. The DAB technique correctly assigned 18 oxidase positive isolates (seven genera), 26 oxidase negative isolates (eight genera), and the organisms present in the two freshly positive blood cultures to their appropriate oxidase reactivity as defined by the standard macroscopic TPD technique. The cytochemical reaction seems to be a reliable indicator of the macroscopic oxidase test. It permits determination of oxidase reactivity at an early stage in the assessment of clinical material when infecting organisms can be demonstrated by microscopy. Further development of this and related cytochemical techniques could permit the provision of microbiological information that would be relevant to patient management, well in advance of conventional techniques.

Evaluation of the activity of chemically identified enteric neurons through the histochemical demonstration of cytochrome oxidase

The measurement of the density of the reaction product produced by the histochemical demonstration of cytochrome oxidase activity provides a method for the visual identification of physiologically active enteric neurons. The current study utilized the cytochrome oxidase technique in order to evaluate the metabolic history of neurons in different regions of the bowel and in chemically identified types of neuron. In addition, the effect of drugs or neurotoxins commonly used in the immunocytochemical identification of enteric neuronal phenotypes was also analyzed. Cytochrome oxidase activity was visualized with a blue-black reaction product resulting from the cobalt-intensified oxidation of 3,3'-diaminobenzidine. Peptides or 5-hydroxytryptamine (5-HT) were localized with biotinylated secondary antibodies and alkaline phosphatase-labeled avidin. Bound avidin or endogenous alkaline phosphatase was visualized with a red reaction product in the presence or absence, respectively, of levamisole. Use of measured without interference from a simultaneously demonstrated histo- or immunochemical marker. A multi-peptidergic class of cholinergic submucosal secretomotor neuron containing neuropeptide Y (NPY) and calcitonin gene related peptide (CGRP) immunoreactivities was found to be less metabolically active than the average of all submucosal neurons. In contrast, a non-cholinergic submucosal secretomotor neuron containing dynorphin (which is also known to contain vasoactive intestinal peptide) immunoreactivity was more metabolically active than submucosal neurons that do not contain this peptide. On average, submucosal neurons were more metabolically active than those of the myenteric plexus, and levels of metabolic activity in the myenteric plexus were found to be higher in the duodenum and the cecum than in the jejunum-ileum or colon. Myenteric neurons characterized by CGRP or NPY immunoreactivities or by endogenous alkaline phosphatase activity, were all less metabolically active than the average of all neurons in myenteric ganglia. Colchicine, which stimulates intestinal motility, was observed to increase cytochrome oxidase activity in enteric neurons, suggesting that an effect on the enteric nervous system contributes to its action on the bowel. The neurotoxins, 6-hydroxydopamine and 5,7-dihydroxytryptamine (5,7-DHT) were each found to stimulate neuronal metabolic activity. 5,7-DHT appeared to activate excitatory subtypes of 5-HT receptor since its effects were blocked or mimicked by compounds that act as antagonists or agonists, respectively, at these receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Microspectrophotometric quantitation of the diaminobenzidine reaction for histochemical demonstration of cytochrome oxidase activity.

Polyacrylamide models in which an extract of cattle heart mitochondria was incorporated, as well as cryostat sections of tongue muscle and epithelium, were used to set up the conditions under which the histochemical reaction for the demonstration of cytochrome oxidase can be quantitated. Using diaminobenzidine in a concentration of 5.5 mM, cytochrome C in a fixed concentration of 76 micron and keeping the incubation medium away from direct light action, enzyme activity can be evaluated by means of direct microphotometry on tissue sections. Each biologic model requires previous individual determination of the measurement limits. These limits can be readily established by using a small chamber for the incubation medium, which can be placed in the microphotometer, allowing the reaction rate to be following using a single section.

On the question of the ultrastructure and composition of corpus intra cristam after local methylcholanthrene application

The study deals with the morphology and composition of the corpora intra cristam in the epidermis of mice after methylcholanthrene application. The bodies appear in all investigated samples 3 weeks after initiation of treatment. In the initial phase, they are probably not always surrounded by a membrane. The limiting membrane is often jaggedly extended, a fact which probably can be explained by the participation of several cristae. Disintegration of the corpus intra cristam-bearing mitochondria and the possibly extramitochondrial appearance of these bodies are discussed. In order to investigate the chemical composition of corpora intra cristam, extraction with chloroform—methanol and with hexane was performed; moreover, pronase digestion and the demonstration of cytochrome oxidase activity was carried out. It was concluded that the investigated intramitochondrial bodies are in all probability composed of a protein with associated phospholipids. Furthermore, it was possible to demonstrate cytochrome oxidase activity mainly in the peripheral zone of the inclusions, which points to the participation of mitochondrial cristae in the formation of the corpora intra cristam.

Demonstration of cytochrome oxidase activity with diaminobenzidine. A biochemical and electron microscopic study.

The relationship between cytochrome oxidase activity assayed biochemically with 3,3'-diaminobenzidine (DAB) as substrate and the resulting electron microscopic staining reaction has been investigated. Mitochondria isolated from both fresh and perfusion-fixed (1% glutaraldehyde and phosphate buffer for 1-2 min) rat liver showed a significant increase in oxygen consumption, as measured by an oxygen electrode, after addition of DAB and were further stimulated by adding cytochrome c. 2,4-Dinitrophenol and antimycin had no effect on the oxidation of DAB; cyanide and azide completely inhibited DAB oxidation, resulting in the absence of any mitochondrial staining reaction. Already after incubation for only 2 min mitochondria exhibited electron-dense deposits on the surface of the inner mitochondrial membrane away from the matrix. The results indicate that DAB donates electrons at the cytochrome c level to the electron transfer system of the respiratory chain and demonstrate the direct relationship between the electron microscopic staining reaction and cytochrome oxidase activity.

The use of diaminobenzidine (DAB) for the histochemical demonstration of cytochrome oxidase activity in unfixed plant tissues.

Cytochrome oxidase activity was demonstrated in unfixed root segments from Lupinus albus at the ultrastructural level using the osmiophilic reagent 3,3′-diaminobenzidine (DAB). Precipitate, the formation of which was completely inhibited by 0.01 M KCN, and observed almost entirely on mitochondrial cristae, is considered to be produced by cytochrome oxidase activity. Heterogeneity of mitochondria as to the intensity of the reaction in the same cell could not be established with certainity. However, mitochondria of the root tip cells and cells belonging to the plerome consistently did not show histochemically demonstrable cytochrome oxidase activity.

Comparison of the ultrastructural demonstration of cytochrome oxidase activity with three bis(phenylenediamines).

Three bis(phenylenediamines) are compared in formaldehyde-fixed rat liver and rat heart. Diaminobenzidine (DAB) demonstrated cytochrome oxidase on mitochondrial cristae, BAXD demonstrated both mitochondrial cytochrome oxidase and a terminal oxidase in endoplasmic reticulum and sarcoplasmic reticulum and BED demonstrated a terminal oxidase only on endoplasmic reticulum and sarcoplasmic reticulum.

Cytochemistry of Membrane-Associated Cytochrome Oxidase in Azotobacter Vinelandii

Young, vegetative cells of the nitrogen-fixing bacterium, Azotobacter vinelandii, contain invaginations of the cytoplasmic membrane. These typical procaryotic cells are characterized by the absence of both a nuclear membrane and organelles such as Golgi bodies and mitochondria (Fig. 1). Although mitochondria are the organelles responsible for electron transport and respiratory operations in eucaryotic organisms, the electron transport function of procaryotic cells is attributed to the membranous structures. In the present study an attempt was made to visualize the site of cytochrome oxidase activity using methods of cytochemistry and electron microscopy.The classical method for the demonstration of cytochrome oxidase activity is the Nadi reaction; the two reagents of this reaction are 1-naphthol and p-phenylenediamine. A substituted p-phenylenediamine, N-benzyl-p-phenylenediamine (BPDA), was used successfully by Seligman et al in the demonstration of cytochrome oxidase activity. This reagent reacts readily with 1-naphthol to form an osmiophilic quinoneimine group resulting in the transfer of electrons to cytochrome c which is subsequently oxidized by cytochrome oxidase. The quinoneimine group reacts with OSO4 to yield an electron dense pigment at the site of cytochrome oxidase activity.

Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB).

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.

Cytochrome oxidase and succinoxidase activity of Limulus gill cartilage

It has been shown that homogenates of gill cartilage prepared from young (2–3 in. long) specimens of Limulus polyphemus, the horseshoe crab, are capable of oxidizing reduced cytochrome c. This activity is inhibited by 10 −4 M cyanide and azide. This marks the first demonstration of cytochrome oxidase activity in cartilage from any source. Such homogenates in the presence of added cytochrome c also mediate hydroquinone oxidation via cytochrome oxidase. Q O 2 values between 6 and 10 were obtained. Oxygen uptake was also inhibited by 10 −4 M cyanide and azide. The presence of malonate-sensitive succinic dehydrogenase and a succinoxidase complex in the gill cartilage homogenates was established.

Oxidative Metabolism of Cartilage

ALTHOUGH utilization of oxygen by mature cartilage is very low, with Qo2 values ranging between 0 and 0.681–5, studies by Rosenthal, Bowie, and Wagoner6 and Boyd and Neuman7 have shown that embryonic cartilage utilizes significantly greater amounts of oxygen (Qo2, 2.2). Paradoxically, although dehydrogenases customarily mediated via cytochrome oxidase (that is, succinic and isocitric dehydrogenases) have been identified in both mature and embryonic cartilage1,8, until now the biochemical demonstration of cytochrome oxidase activity in cartilage had not been accomplished.