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

Summary 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.     

The use of tetrazolium salts in the histochemical demonstration of succinic dehydrogenase activity in plant tissues

Summary Succinic dehydrogenase activity was determined in fresh or cryostat sections of tissues from Allium cepa, Vicia faba, Pisum sativum and Helianthus tuberosus using different tetrazolium salts as electron accepters. In 10 fresh or frozen sections a reaction was obtained with TNBT, NBT, MTT, and INT but not with NT, BT or TTC. In contrast a reaction was obtained with each of the tetrazolium salts in 50–150 fresh or frozen sections. The observed differences in the abilities of the tetrazolium salts to demonstrate succinic dehydrogenase activity are discussed.The sites of acceptance of electrons from the electron transport pathway in plant cells by the tetrazolium salts has been demonstrated cytochemically, and shown to differ from those observed in animal cells in that unlike animal cells, there is no apparent acceptance of electrons by MTT and INT from cytochrome C₁-C region of the pathway.     

Partial inactivation of cytochrome c oxidase by nonpolar mercurial reagents

Purified beef heart cytochrome c oxidase is inactivated to the extent of 35 to 50% by the nonpolar mercurial reagents mercuric chloride and ethylmercuric chloride. The inactivation is complete within 5 min. In titrations of activity, the plateau level of inactivation is attained at added ethylmercuric chloride:heme a ratios of about 1:1. Up to 3 mercury atoms/heme a are bound to the oxidase, although only the first of these affects its enzymatic activity. Incubation of the ethylmercury-modified oxidase with sulfhydryl compounds reverses the inactivation, with 2,3-dimercaptopropanol being most effective of the reagents tested. Spectrophotometric and polarographic assays of enzymatic activity show that Km values for the native and the ethylmercury-modified enzymes are practically indistinguishable, and that the partial inactivation observed for the latter is reflected exclusively in a lower value of Vmax compared to that of the native enzyme. Based on these results, we propose that ethylmercuric chloride reacts with a single crucial--SH group per heme a, and that electron transfer processes in the modified product are partially inhibited.     

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.     

Studies on the oligomeric state of isolated cytochrome oxidase using cross-linking reagents

(1) Sucrose gradient centrifugation of cytochrome oxidase in the presence of Triton X-100 gave one slowly sedimenting green band. After cross-linking with dithiobis(succinimidylpropionate) (DSP), two green bands were observed, one sedimenting like the control and the other one more rapidly. Only the slowly sedimenting band was observed if the cross-linker was cleaved by dithiothreitol before centrifugation. (2) The rapidly sedimenting band in the Triton-containing sucrose gradient is probably the internally cross-linked dimer of cytochrome oxidase; the one sedimenting slowly is the monomeric enzyme. (3) Cross-linking with DSP after monomerization yields a small fraction of internally cross-linked dimers in addition to the internally cross-linked monomers. Under similar conditions, but using the shorter cross-linker disuccinimidyl tartarate (DST), no dimers are detected. (4) Both DSP and DST cross-link the dimeric enzyme so that it could no longer be monomerized by centrifugation in Triton, unless the cross-link is cleaved. (5) Polypeptide analysis using two-dimensional gel electrophoresis of cross-linked dimers and monomers suggest that subunit VIb is involved in intermonomeric cross-linking of dimeric enzyme by DSP.     

Endogenous cytochrome c and cytochrome oxidase in rat and mouse kidney: light microscopic histochemical study.

Activity of cytochrome c oxidase and the level of endogenous cytochrome c were investigated light microscopically in adult rat and mouse kidney by incubating unfixed frozen sections with diaminobenzidine (DAB) in the absence or presence of exogenous cytochrome c. The results suggest that DAB staining intensity mainly reflects the local density of mitochondria and only occasionally visualizes the differences in cytochrome oxidase activity and/or endogenous cytochrome c content. Most intense reaction was observed in proximal and distal tubules both in rat and mouse. Finer differentiation of reactivity in particular nephron segments and interspecies differences between rat and mouse kidney are also described.     

Comparison of kinetic and end-point microdensitometry for the direct quantitative histochemical assessment of cytochrome oxidase activity

Synopsis Cytochrome oxidase activity has been assessed by a method of kinetic microdensitometry which involves applying tissue sections to gel films containing phenylamine substrates and measuring the rate of azine dye production by continuously recording the rate of change in extinction. Optimum conditions for the technique were defined, and the results compared with those obtained by conventional end-point microdensitometry in which sections are incubated in histochemical substrate solutions and azine dye production estimated by a single measurement of extinction at the end of the incubation period. When compared with biochemically-determined enzyme activity, kinetic microdensitometry gave a better index of the proportionate activity of cytochrome oxidase in various normal tissues than did end-point microdensitometry. In addition, the degree of inhibition of cytochrome oxidase activity in tissues removed from cyanide-poisoned animals was assessed more reliably by kinetic microdensitometry than by end-point measurements. With end-point microdensitometry, the reaction is non-linear over the comparatively long incubation times required and there is also a spontaneous reactivation of cyanide-inhibited cytochrome oxidase during incubation and thus a progressively increased rate of substrate utilization. In contrast, with kinetic microdensitometry the initial linear reaction rate is measured before significant reactivation occurs. Kinetic microdensitometry can be used for direct dynamic quantitation of enzyme activity in tissues or cells; it may be a valuable technique for quantitative histochemical confirmation or extension of biochemical studies; and it appears to be a reliable direct quantitative histochemical method for investigatingin vivo inhibition of enzyme activity, where spontaneous reactivation of the enzyme-inhibitor complex may occur.     

A near-infrared investigation of cytochrome c oxidase in higher plant mitochondria

Optical features of cytochrome c oxidase in potato mitochondria have been characterized in the near-ir region. In order to discriminate the respective properties of the various redox centers, the redox state was monitored from free and inhibited, bound species. Appropriate comparisons singled out difference spectra which can be attributed specifically to CuA and CuB. The CuA difference spectrum (red-ox) exhibits a negative band centered at 812 nm and, analogous to its mammalian counterpart, the so-called 830-nm band (delta epsilon red/ox = -2.0 mM-1 cm-1). The unusual difference spectrum (red-ox) assigned to CuB is characterized by a broad positive band also centered at 812 nm with an extinction coefficient of delta epsilon red/ox = 4.3 mM-1 cm-1.     

Preparation and structure-activity relationship of reagents for cytochrome oxidase activity: potential for light and electron microscopy

Summary The syntheses of a series of p-substituted aromatic diamines and some representative thioureido- and mercapto-naphthols are described. Their cytochemical behavior in the Nadi reaction using fresh frozen sections of rat heart was studied in an attempt to learn more about the structural requirements of the Nadi reaction for cytochrome oxidase.The lipid solubility of the dyes formed from either N,N-dimethyl-p-phenylenediamine or ADN (4-amino-1-N,N-dimethylnaphthylamine) with 1-naphthol can be decreased by substituting hydrophilic hydroxyethyl or dihydroxypropyl side chains for one or more of the methyl groups in these reagents. However, these dyes diffuse readily, fade rapidly and are too soluble in water as well as lipid, which renders them unsuitable for light and electron microscopy.Burstone's reagent PPD (N-phenyl-p-phenylenediamine) and ADN (4-amino-1-N,N-dimethylnaphthylamine) are capable of undergoing self condensation either alone or in the presence of an unreactive naphthol to give colored indamines which osmicate readily. Blocking either the 4-position of N-phenyl-p-phenylenediamine, the secondary amino group, or both of these positions, with a methyl group did not prevent indamine formation.N-Benzyl-, N-4-methylbenzyl- and N-4-methoxybenzyl-p-phenylenediamine are good reagents for the demonstration of cytochrome oxidase activity in the Nadi reaction with light microscopy. They produce blue indoaniline dyes with 1-naphthols and do not self condense to form highly colored pigments. These indoanilines are osmiophilic eliminating the necessity for introducing osmiophilic groups for electron microscopic studies. Their drawback in electron microscopy is in the droplet nature of the deposit.Incorporating a carbonyl or carboxyl function into the 2-position or a bulky substituent into the 5-position of 1-naphthol prevented indoaniline formation.A new method for the cytochemical demonstration of cytochrome oxidase is presented utilizing a new reagent N,N-bis(p-aminophenyl)-1,3 xylylenediamine (XIII) (BAXD). This reagent is polymerized to an insoluble osmiophilic polymer distributed in non-droplet form, thus providing a useful method for demonstrating cytochrome oxidase activity in light and electron microscopy.This investigation was supported by a research grant (CA-02478) from the National Cancer Institute, U.S. Public Health Service, Bethesda, Maryland. Presented (in part) as a Presidential Address (AMS) at the Third International Congress of Histo- and Cytochemistry on August 19, 1968 in New York, N.Y. Acknowledgement is due Lionel, Katzoff for preparation of the thiolnaphthols XXXV and XXXVI, and to Norberto I. Schinitman for the osmium analysis.     

The use of the fluorescent probe 8-anilino-1-naphthalene sulphonic acid (ANS) as a histochemical stain in plant tissues

Abstract. The fluorescent probe 8-anilino-l-naphthalene sulphonic acid (ANS) has been evaluated as a histochemical stain for plant tissues. The wide specificity of the compound for hydrophobic binding sites limits its analytical use, but renders it of considerable value as a general fluorescent stain for use in epi-illumination fluorescence microscopy. Used in this way it is analogous to the light-microscope stain toluidine blue. ANS has also been found to be a sensitive vascular tracer.