Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.9.3.1 (cytochrome oxidase)
8,822 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Diaminobenzidine, DAB, was applied to segments of aerobically and anaerobically grown coleoptiles of rice, Oryza sativa L., with the object of studying the location of cytochrome oxidase at the electron-microscope level. A specific staining of mitochondrial cristae and inner membrane was obtained, with no reaction in other organelles; with extended periods of incubation, the reaction product filled the mitochondria completely. In anaerobically grown coleoptiles, the reaction was much slower and the difference was particularly marked in vascular bundle companion cells and parenchyma, which gave the strongest reaction in aerobic tissue, but in the anaerobic stained even less than the cortical parenchyma. The reaction was inhibited by boiling and slowed very much by lowering of the incubation temperature from 27 to 4 degrees C. This indicated the involvement of an enzymic reaction and cyanide inhibition indicated that a haem enzyme was involved. The catalase inhibitor aminotriazole did not inhibit DAB oxidation. Nevertheless the specificity of the reaction for cytochrome oxidase must be questioned, because preheating of the tissue to 60 degrees C before incubation, which would be expected to destroy cytochrome oxidase activity, failed to decrease the oxidation, at least in aerobically grown coleoptiles. It is concluded that DAB is oxidized in the rice coleoptile tissue by a cytochrome system, and the development of this system is inhibited by anaerobiosis, but the oxidation cannot be claimed to represent cytochrome oxidase activity exclusively. Perhaps other autoxidizable, more heat-stable cytochromes participate in the reaction.
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PMID:The reaction of mitochondria in the coleoptiles of rice (Oryza sativa L.) with diaminobenzidine. 4 31

Direct histocytochemical staining methods on undisrupted tissues, stabilized by chemical fixation, potentially offer perhaps the most reliable approach to the study of the enzymes of the cell with relation to its ultrastructure. The atoms which, for the most part, comprise the biomacromolecules and enzymes of cells and tissues contribute little to their inherent electron opacity or ability to scatter electrons differentially. The latter property of a substance is responsible for its observation with the electron microscope. Since the introduction of osmiophilic reagents into cytochemistry (HANKER et al. 1964), the selective deposition of relatively large amounts of polymeric osmium black reaction products at the subcellular sites of insoluble or immobilized enzymes or biomacromolecules has facilitated their demonstration with the light and electron microscopes. Perhaps the most widely employed osmiophilic reagent in histocytochemistry has been DAB which was introduced by GRAHAM and KARNOVSKY (1966a, b). Although it receives its widest use for demonstrating the sites to which the exogenous ultrastructural tracer horseradish peroxidase (HRP) is transported in vertebrate tissues, it is also widely employed for the demonstration of catalase in peroxisomes with the media of FAHIMI (1969) or of NOVIKOFF and GOLDFISCHER (1969), and for the demonstration of cytochrome oxidase with the medium of SELIGMAN et al. (1968a). The importance of this reagent lies in its ability to undergo oxidative polymerization forming an insoluble osmiophilic melanin-like product (HANKER et al. 1972a) which comforms well to ultrastructure, at the sites of enzymic or nonenzyme proteins which catalyze its oxidation. In the past few years, studies in our laboratory have shown that a rational approach to the histocytochemical demonstration of enzymes could be devised. It is based on the selective deposition of transition metal compounds at the sites of enzymes that resemble hemoproteins in their ability to catalyze the oxidative polymerization of DAB. The most useful of these compounds, cupric ferrocyanide (Hatchett's brown) was also introduced into cytochemistry by Karnovsky's laboratory (KARNOVSKY 1964; KARNOVSKY and ROOTS 1974). By the use of natural substrates, when available, or synthetic substrates which liberate or form a reducing agent at the sites of the enzymatic activity, many diverse types of enzymes have been demonstrated by methods depending on this principle known as catalytic osmiophilic polymer generation. DAB has probably been the most useful histocytochemical reagent of the past decade. Yet its borderline carcinogenicity and the frequent interruption of a supply of good quality DAB have encouraged research into a substitute reagent. A new substitute for DAB has resulted from the study of artificial melanins in our laboratory for several years. It consists of a mixture of p-phenylenediamine and pyrocatechol and is much better than DAB for the demonstration of HRP used as a cytochemical tracer...
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PMID:Osmiophilic reagents in electronmicroscopic histocytochemistry. 9 99

The ultrastructural localization of the activities of two enzyme systems in the culture forms of Leishmania donovani was shown by means of the diaminobenzidine techniques. The consistent deposition of electron dense reaction product of DAB oxidation without H2O2 in the kinetoplast and mitochondrial cristae and membranes was taken as evidence of the presence of cytochrome oxidase activity and cytochrome c. In the presence of H2O2, a more intense DAB oxidation was attributed to the activity of a peroxidase, possibly cytochrome c peroxidase. Mitochondrial and kinetoplast reactions to DAB were completely inhibited by KCN, methanol-nitroprusside, and by heating to 50 degrees C for 10 min. On the other hand, no inhibitory effect was observed with 100 mM 3-amino-1,2,4-triazole. Under all conditions of incubation tested, the microbodies were completely unreactive to DAB staining, which was utilized as the basis for their identification. These organelles are rounded, moderately electron-opaque bodies with a finely granular matrix and fine tubules or cores and are limited by a single membrane. Under normal staining method, the microbodies were indistinguishable from the rounded sections of mitochondria.
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PMID:Ultrastructural localization of diaminobenzidine reactivity in leishmania donovani promastigotes. 19 23

Intracellular amastigotes of Leishmania donovani obtained from spleens of infected hamsters were studied by means of the diaminobenzidine technique for the presence of cytochromes and the activities of cytochrome oxidase and peroxidase. In the absence of H2O2, the oxidation of DAB, evidenced by electron-dense deposits localized on the cristae, inclusions, and enveloping membranes of the mitochondria and kinetoplast, revealed the activity of the cytochrome oxidase and the presence of the cytochromes. The increased deposition of DAB oxidation especially on the enveloping membranes in the presence of H2O2 suggests the activity of a peroxidase, probably cytochrome c peroxidase.
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PMID:Leishmania donovani: ultrastructural localization of diaminobenzidine reactivity in the amastigotes. 21 7

The cytochrome oxidase activity of rat dorsal root ganglion cells was cytochemically and quantitatively measured. The 100 microns thick tissue slices fixed for 10 min were incubated in a DAB medium for 0, 30, 60, and 90 min at 37 degrees C, and electron-dense deposit areas within the mitochondrial intermembrane-intracristal spaces were measured with a computer-controlled image analyzer. The activity was expressed as deposit accumulation rate filling the mitochondrial space, (1 unit corresponds to the deposit filling 100% of the mitochondrial space per hour). Three different activities of the enzyme in either large pale cells or small dark cells of sensory neurons could be distinguished, based on the quantitative analysis, as large cells with intense (0.83 units), intermediate (0.38), and weak activity (0.22), and small cells with the same degrees of activity (1.04, 0.35, and 0.11, respectively). The results indicate that accumulation rate measurements of reaction product may be useful to quantitatively present the cytochrome oxidase reactivity of mitochondria, and that the degree of enzyme activity may contribute to the identification of functional differences in sensory neurons.
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PMID:Quantitative cytochemical studies of cytochrome oxidase activity in rat dorsal root ganglion cells. 217 51

The turnover of total mitochondrial proteins and cytochrome oxidase in the livers of rats administered with 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) has been determined. The incorporation of [14C]bicarbonate revealed a half-life of 3.1 days in control and 6 to 9 days in azodye administered animals for whole mitochondrial proteins. The incorporation of [35S]methionine yielded t1/2 values of 8.5 days and 15.4 days, respectively. The t1/2 of cytochrome oxidase, 10.8 days for control and 19.3 days for 2-Me-DAB-treated animals, indicated that the delay in the decay of the enzyme was of the same order as that of whole mitochondria. Short term incorporation revealed that the administration of the azodye stimulated the synthesis of the enzyme. Mitochondria isolated from azodye-administered animals appeared less susceptible to lysosomal proteolysis. Also, azodye administration seemed to impair the ability of lysosomes to degrade mitochondria.
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PMID:Effect of administration of 2-methyl-4-dimethylaminoazobenzene on the half-lives of rat liver mitochondria and cytochrome oxidase. 298 8

Basically the DAB-technique localizes 3 enzymes, i.e. peroxidase, catalase, and cytochrome oxidase, but also pseudoperoxidatic activity of hemeenzymes (hemoglobin, myoglobin, etc.). Although at the ultrastructural level, i.e. in cytochemistry, the appropriate conditions for specific identification of each of these enzymatic activities have been extensively studied and reported in the literature, the subject remains open to investigation. In light microscopy DAB staining has been less thoroughly studied. Since DAB histochemistry might have practical interest in daily diagnostic pathology, it appeared worthwhile to work out a method convenient for paraffin embedded tissues. The method consisted of a prolonged incubation 48 h) of small tissue blocks, which had been prefixed for 1 h in 4% formaldehyde. Dehydration and rehydration occurred in graded ethanols; counterstain was obtained by toluidine blue. Although further experiments are needed to specify the physico-chemical conditions for the three enzymatic activities, the results are morphologically superior to that of frozen sections.
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PMID:Diaminobenzidine histochemistry in light microscopy. 617 Nov 31

In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specificity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15-30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylte buffer pH 6.5 and 0.02% H2O2. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) buffer at pH 10.5 and 0.15% H2O2. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.
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PMID:Selective cytochemical localization of peroxidase, cytochrome oxidase and catalase in rat liver with 3,3'-diaminobenzidine. 626 82

For demonstration of the local cytochrome oxidase activity, coronal sections through the frontal cortex of four Sprague-Dawley rats were incubated with DAB medium after Seligman et al. (1968). The pattern of distribution of the DAB reaction product (DAB-RP) was measured at a wavelength of 460 nm. Cryostat sections were scanned with a microdensitometer. The cytochrome reaction product was accumulated in the laminae I and IV. A comparative morphometric study of mitochondrial profiles in ultra-thin sections through equivalent cortical regions showed an approximately similar distribution in percentage of areal density of the mitochondria labeled by DAB-RP. On average 30% of the mitochondrial profiles were labeled with DAB-RP which was localized in the intracristate spaces and the outer mitochondrial compartment. The marked mitochondria mainly occurred in dendrites. Furthermore, the mean and median value of the size distribution of marked mitochondrial profiles was shifted to greater values when compared with non-marked ones.
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PMID:Local cytochrome oxidase activity in the cerebral cortex of the rat, histochemically detected with the DAB-method: a micro-densitometric and electron microscope study. 632 2

Thick biological specimens prepared as whole mount cultured cells stained with histochemical reactions, such as thiamine pyrophosphatase, glucose-6-phosphatase, cytochrome oxidase, acid phosphatase, DAB reactions demonstrating specific cell organelles such as Golgi apparatus, endoplasmic reticulum, mitochondria, lysosomes, peroxisomes and pinocytotic vesicles, were observed by ultrahigh voltage electron microscopy at accelerating voltages of 400-1000 kV producing stereo-pairs. As a result, those cell organelles were observed 3-dimensionally and the relative relationships between these organelles demonstrated.
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PMID:Three dimensional observation of whole mount cultured cells stained with histochemical reactions by ultrahigh voltage electron microscopy. 853 71


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