The Cytochemical Localization of Oxidative Enzymes : I. Diphosphopyridine Nucleotide Diaphorase and Triphosphopyridine Nucleotide Diaphorase

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.     

The cytochemical localization of oxidative enzymes. II. Pyridine nucleotide-linked dehydrogenases

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.     

The Cytochemical Localization of Oxidative Enzymes : II. Pyridine Nucleotide-Linked Dehydrogenases

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.     

The Histochemical Localization of Triphosphopyridine Nucleotide Diaphorase

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.     

Oxidation of reduced triphosphopyridine nucleotide by submitochondrial particles from beef heart

Submitochondrial particles prepared from beef heart are capable of oxidizing TPNH, in the absence of added DPN, at a rate of approximately 50 nmoles/min × mg protein at 30°. TPNH oxidation by these particles occurs through the respiratory chain as evidenced from TPNH-induced reduction of the cytochromes and the inhibitory effects of rotenone, piericidin A, amytal, antimycin A and cyanide. The latter studies have indicated that the site of TPNH interaction with the respiratory chain is on the substrate side of the rotenone-piericidin block and close to that of DPNH.     

Microsomal electron transport reactions. II. The use of reduced triphosphopyridine nucleotide and-or reduced diphosphopyridine nucleotide for the oxidative N-demethylation of aminopyrine and other drug substrates

The ratio of formaldehyde formed to TPNH oxidized during aminopyrine oxidative demethylation as catalyzed by rabbit liver microsomes was found to be about 0.5. This is less than the expected 1:1 ratio for a mixed function oxidase reaction and may reflect the oxidation of TPNH by other reactions. Similar results were obtained when measuring the oxidative demethylation of codeine and ethylmorphine. In all cases the addition of DPNH significantly increased the yield of formaldehyde formed in the presence of TPNH. The stimulatory effect of DPNH was a linear function of the DPNH concentration added until the initial concentrations of DPNH and TPNH were equal. Increasing the DPNH concentration above a DPNH:TPNH ratio of 1:1 had no further effect upon the final concentration of formaldehyde formed. This observation, as well as the inhibition of DPNH-supported aminopyrine metabolism by TPN⁺, argue against the role of a transhydrogenase mechanism for the DPNH effect. The rate of DPNH oxidation catalyzed by liver microsome was also observed to increase markedly in the presence of TPNH.     

Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Cofactor Requirements

The rates of oxidation of ent-kaur-16-ene to ent-kaur-16-en-19-ol, ent-kaur-16-en-19-al, ent-kaur-16-en-19-oic acid, and ent-kaur-16-en-7alpha-ol-19-oic acid are maximal in microsomes prepared from the endosperm of immature Marah macrocarpus seeds in which the cotyledons are approximately one-half the overall length of the seed. The supernatant fraction remaining from the preparation of the microsomes contains factors which stimulate the rates of oxidation catalyzed by the microsomes. Added TPNH is more effective than added DPNH in meeting the requirement for reduced pyridine nucleotide. A mixture of DPNH, ATP, and TPN(+) is much more effective than DPNH alone. Experiments with 2,4-dinitrophenol as a selective inhibitor indicate that the ATP-stimulated synthesis of TPNH which occurs in these microsomes in the presence of this mixture of coenzymes provide TPNH for use in the mixed function oxidations. Relatively low concentrations of DPNH and TPNH together are much more effective than either alone at equivalent concentration. This is consistent with the involvement of two pathways of electron transfer associated with the mixed function oxidations, one of which preferentially utilizes TPNH and the other favoring DPNH. FAD added to microsomes at an optimal concentration of about 10 mum in the presence of TPNH stimulates the rate of the oxidations; higher concentrations are inhibitory. FMN by itself does not produce this stimulation. However, FMN and FAD added together at low concentrations (0.5 mum each) have approximately the same effectiveness as FAD alone at 10 mum. This suggests a role for both flavin nucleotides in the normal electron transfer pathways associated with these oxidations. Some of the stimulatory properties of the supernatant fraction may be accounted for by its content of reduced pyridine nucleotides, FAD, and FMN; the concentrations of FAD and FMN were determined to be 1.1 mum and 0.4 mum, respectively. However, the effects of the supernatant fraction are not completely explained by its content of these coenzymes since other experiments indicate the presence of a heat-labile, nondialyzable stimulatory factor(s) in the supernatant fraction in addition to heat-stable, dialyzable fractors.     

Skeletal muscle fibres show NADPH diaphorase activity associated with mitochondria, the sarcoplasmic reticulum and the NOS-1-containing sarcolemma

The subcellular appearance of NADPH diaphorase activity in different rat skeletal muscles has been analyzed. Both a sarcolemma-associated as well as a non-sarcolemma-associated NADPH diaphorase-dependent generation of formazan was observed. The sarcolemma-associated NADPH diaphorase staining appeared regularly in two manifestations: one observed in longitudinal sections as dotted costameres at the cell surface which accordingly appeared in transversal sections as rings surrounding the myofibre surface. At this site, nitric oxide synthase (NOS)-1 was located. The second sarcolemma-associated site of NADPH diaphorase staining was found as bundles of longitudinal-orientated stripes of hitherto unidentified origin. The non-sarcolemma-associated production of formazan was likewise manifested at two sites: the first was found regularly in longitudinal sections as intense sarcomere-like striations occurring parallel to the I-bands and indicating mitochondria. The second non-sarcolemma-associated NADPH diaphorase staining was realized as fine longitudinal filaments of variable occurrence connecting the mitochondria and presumably belonging to the sarcoplasmic reticulum. Attempts to identify single NADPH diaphorase(s) existing in skeletal muscles by incubation with specific inhibitors failed but showed the presence of two different subpopulations of NADPH diaphorases in myofibres: a urea-resistant fraction in the sarcolemma region containing NOS-1 and a non-sarcolemma-associated, urea-sensitive fraction depleted of NOS-1.     

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine.

Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with NG-nitro-L-arginine, a NOS inhibitor, or L-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.     

PYRIDINE NUCLEOTIDE-LINKED REACTIONS OF PSEUDOMONAS NATRIEGENS.

Eagon, R. G. (University of Georgia, Athens). Pyridine nucleotide-linked reactions of Pseudomonas natriegens. J. Bacteriol. 84:819-821. 1962-The observation that Pseudomonas natriegens utilizes the Embden-Meyerhof pathway and the hexose monophosphate-pentose cycle only very slightly, even though the necessary enzymes are present, was explained by the existence of a sluggish system for the oxidation of reduced triphosphopyridine nucleotide (TPNH). Pyridine nucleotide transhydrogenase could not be detected in cell-free extracts. A very active system for the oxidation of reduced diphosphopyridine nucleotide (DPNH) was observed. Thus, since lactic acid is a major end product of glucose dissimilation and since the lactic dehydrogenase of P. natriegens does not utilize DPNH as cofactor, the Embden-Meyerhof pathway apparently operates aerobically by direct oxidation of DPNH, presumably by coupling with the terminal oxidase system rather than by coupling to synthetic reactions requiring DPNH as cofactor. A TPNH-specific glutathione reductase was detected which was inhibited by adenosine-2'-monophosphate.     

Respiratory Mechanisms in the Aroid Spadix

The flowers of Skunk-cabbage (Symplocarpus foetidus), like thespadix tissues of other Aroids, have a rapid, carbon monoxideand cyanide (HCN) resistant respiration; oxygen uptake is independentof the oxygen partial pressure over a wide range. Cell fractionswere isolated by differential centrifugation and their oxidativeactivities studied. Oxidation of succinate and citrate by mitochondriacan be inhibited 50 to 60 per cent. by 1 X 10^–3 M. HCN,and antimycin A (AA) causes partial inhibitions. An active mitochondrialcytochrome-c oxidase is present, and it shows a typical sensitivityto cyanide. The mitochondria possess an active reduced diphosphopyridine-nucleotide(DPNH) oxidase system, which is inhibited roughly 80 per cent.by 1 X 10^–3 M. HCN and 1.7 µg./ml. AA. The microsomalDPNH oxidase, which is less sensitive to inhibitors, is lessactive per gramme of tissue than that on the mitochondria. Thefinal supernatant shows little DPNH oxidase. With all fractions,reduced triphosphopyridine nucleotide (TPNH) is oxidized muchmore slowly than DPNH. DPNH-cyto-chrome-c reductase activitywas measured; the mitochondrial system is partially blockedby AA, whereas the microsomal activity is AA-insensitive. Spectro-photometricexamination of a preparation of solubilized mitochondria showedthat cytochromes a, b, and c are present. The results are discussedwith reference to the pathway and localization of hydrogen andelectron transport in the Aroid spadix.     

Reduction of tetrazolium salts catalyzed by soluble diaphorase in rat liver and embryos of Vicia faba

Summary The presence of two diaphorases has been shown in rat liver and embryos of Vicia fdba. One of these, the NAD(P)H tetrazolium reductase, was firmly bound in the section and was not lost into the incubation medium under conditions of histochemical assay The second diaphorase (soluble diaphorase) was lost from the section into the incubation medium during the first five minutes of incubation. This soluble diaphorase from both rat liver and embryos of V. faba is capable of transferring electrons from NAD(P)H to MTT, INT, NBT and TNBT, but not to tellurite, TTC, BT and NT. The behaviour of the soluble diaphorase in histochemical reactions involving tetrazolium salts as electron acceptors is discussed.     

Co-localization of nitric oxide synthase immunoreactivity and NADPH diaphorase staining in neurons of the guinea-pig intestine

Summary Neuronal nitric oxide synthase (NOS), an enzyme capable of synthesizing nitric oxide, appears to be identical to neuronal NADPH diaphorase. The correlation was examined between NOS immunoreactivity and NADPH diaphorase staining in neurons of the ileum and colon of the guinea-pig. There was a one-to-one correlation between NOS immunoreactivity and NADPH diaphorase staining in all neurons examined; even the relative staining intensities obtained were similar with each technique. To determine whether pharmacological methods could be employed to demonstrate that NADPH diaphorase staining was due to the presence of NOS, tissue was pre-treated with N^G-nitro-l-arginine, a NOS inhibitor, or l-arginine, a natural substrate of NOS. In these experiments on unfixed tissue, it was necessary to use dimethyl thiazolyl tetrazolium instead of nitroblue tetrazolium as the substrate for the NADPH diaphorase histochemical reaction. Neither treatment caused a significant decrease in the level of NADPH diaphorase staining, implying that arginine and NADPH interact at different sites on the enzyme.     

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

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.     

Cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity.

The enzyme 5-formyl tetrahydrofolate cyclodehydrase plays an important role in the conversion of 5-formyl tetrahydrofolate to 5,10-methenyl tetrahydrofolate. A second enzyme, cyclohydrolase, converts 5,10-methenyl tetrahydrofolate to 10-formyl tetrahydrofolate. These folate derivatives play a significant part in the biosynthesis of purines. A method has been devised for the cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity which uses 5-formyl tetrahydrofolate or 5,10-methenyl tetrahydrofolate as substrate respectively, blocking possible interferences by other enzymes, and allows the nonenzymatic reduction of nitro-blue tetrazolium by 5,10-methenyl tetrahydrofolate formed by the action of the cyclodehydrase on the substrate 5-formyl tetrahydrofolate, and by 10-formyl tetrahydrofolate formed by the action of cyclohydrolase on the substrate 5,10-methenyl tetrahydrofolate, thus revealing intracellular sites of enzyme activity. The methods appear to show only intracellular localization of the blue formazan deposits of reduced tetrazolium. The distribution of positivity in cells of human blood and bone marrow is described.     

Ultrastructural cytochemistry of p-N,N-dimethylamino-beta-phenethylamine (DAPA) oxidation reactions.

The ultracytochemical localization of amine oxidase (AO) activity is demonstrated with a new substrate, p-N,N-dimethylamino-beta-phenethylamine (DAPA). DAPA was designed to yield a stronger reducing agent on oxidation by monoamine oxidase (MAO) than is obtained from the MAO substrate, tryptamine, upon oxidation. Thus MAO and possibly other oxidase(s) can be demonstrated with DAPA and the tetrazolium salt, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT). The latter is a nonosmiophilic tetrazolium salt which is reduced to an osmiophilic formazan. In addition, DAPA itself demonstrates AO activity ultracytochemically with and without BSPT. We speculate that either oxidative polymerization of DAPA or Schiff's base formation with protein after aldehyde formation is responsible for the latter reaction, which is made permanent for ultracytochemical localization by osmication at a later step. DAPA oxidation reaction products are demonstrated in guinea pig kidney, specifically in the endoplasmic reticulum, nuclear envelope and mitochondrial outer compartments and cristae. Differences in reaction product characteristics and localization in relation to formaldehyde fixation and the localization of reaction product in mitochondrial cristae, as well as outer compartments, suggest that DAPA oxidation is mediated through one or more MAOs and possible other oxidases.     

The electron cytochemistry of ubiquinone in mammalian heart,demonstrated with a monotetrazolium salt

Ubiquinone (Coenzyme Q) and similar compounds can be demonstrated in tissue sections by means of a system using three redox couples. The last of these involves the reduction of a tetrazolium salt to its formazan. Using glutaraldebyde fixed blocks of rat heart muscle the final product has been demonstrated, in and on the surface of the mitochondria, as strongly electron opaque deposits 0.1 to 0.3 microns across. These results offer confirmation of biochemical and light microscopical observations on the localisation of ubiquinone. The secondary development reactions employed may be useful for the conversion of compounds other than formazans to electron opaque material.In receipt of a Research Grant from the British Heart Foundation.     

A fluorescent redox dye. Influence of several substrates and electron carriers on the tetrazolium salt-formazan reaction of Ehrlich ascites tumour cells

Summary This study was performed to elaborate the best conditions for measuring the redox activity of Ehrlich ascites tumour cells by using a new tetrazolium salt, cyantolyl tetrazolium chloride (CTC). This tetrazolium salt forms a fluorescent water-insoluble formazan on reduction on the surface of intact vital cells. The influences of fixation and of various substrates and electron carriers on the cellular reduction of CTC were investigated quantitatively using an elution technique. The amount of formazan obtained after incubating vital cells with Meldola Blue as electron carrier was greater than that obtained with Methylene Blue, menadione, 2,6-dichloroindophenol, 1-methoxyphenazine methosulphate or phenazine methosulphate. Using flow cytometry, the formazan production per cell and, after staining the nuclear DNA, the distribution of the redox activity in the cell population can be visualized with satisfactory resolution. We conclude from our findings that dehydrogenases are only partially involved in the reduction of tetrazolium salts by intact cells and that a redox activity, probably related to a cell membrane-bound NAD(P)H—oxidase system, is mainly measured.     

Ultrastructure and enzyme histochemistry of the pancreatic islets in the horse

Summary The characteristic localization of the silver-negative A₂ cells in the central part of the pancreatic islets in the horse offers a good opportunity to study the ultrastructure and histochemistry of this type of islet cell. Electron microscopical analyses revealed that the A₂ cells contained dense spherical granules varying considerably in size. Light and dark A₂ cells were identified. The presence of numerous secretory granules of very low density was the most conspicous feature of the B cells. These cells also showed considerable differences in density. A second type of peripheral islet cell was characterized by a very high content of mitochondria and ribosomes. These small islet cells contained tiny granules and are probably identical with the A₁ cells.Negative reactions for alkaline and acid phosphatases were obtained throughout the islet tissue, while a strong glucose-6-phosphatase activity was displayed by the peripheral cells. The diphosphopyridine and triphosphopyridine nucleotide diaphorase activities were high in the peripheral cells, considerably weaker reactions being noted in the A₂ cells. On the whole there was a low succinic dehydrogenase activity in the islet tissue with a somewhat weaker enzyme staining in the A₂ than in the peripheral cells. The reactions for glucose-6-phosphate dehydrogenase and lactic dehydrogenase were also less pronounced in the A₂ cells than in the intensely reacting peripheral cells.The following abbreviations are used DPN Diphosphopyridine nucleotide - DPND Diphosphopyridine nucleotide diaphorase - DPNH Diphosphopyridine nucleotide, reduced form - G-6-PD Glucose-6-phosphate dehydrogenase - LD Lactic dehydrogenase - MTT 3,5-diphenyl-2-(4,5-dimethylthiazol-2-yl)-tetrazolium bromide - Nitro-BT 2,2-di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - SD Succinic dehydrogenase - TPN Triphosphopyridine nucleotide - TPND Triphosphopyridine nucleotide diaphorase - TPNH Triphosphopyridine nucleotide, reduced form Supported by the Swedish Medical Research Council and the research grant A-5759 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.     

In vitro superoxide activity in the haemolymph of the West Indian leaf cockroach,Blaberus discoidalis

The respiratory burst is an NADPH oxidase-driven reduction of molecular oxygen to superoxide, which can occur in phagocytic cells as part of an antimicrobial defence, and is well documented among the vertebrates. This paper describes a process resembling the respiratory burst, which occurs in the haemolymph and haemocytes of the cockroach, Blaberus discoidalis. The in vitro reduction of nitroblue tetrazolium by superoxide to formazan was measured spectrophotometrically in B. discoidalis haemolymph in response to various immune elicitors. Nitroblue tetrazolium reduction was partly impeded in the presence of superoxide dismutase, a specific antioxidant which converts superoxide to hydrogen peroxide, as well as by chemicals known to inhibit the respiratory burst in vertebrates (trifluoperazine, diphenylene iodonium, and N-ethylmaleimide). This suggests the generation of superoxide anions by haemolymph as part of an immune response. Furthermore, formazan staining of elicitor-treated haemocytes was observed microscopically, with less intense staining in the presence of superoxide dismutase. Finally, respiratory burst inhibitors and superoxide dismutase enhanced the growth of E. coli incubated in whole haemolymph, implying a role for haemolymph-derived superoxide in antibacterial defence.