scholarly journals CYTOCHEMICAL LOCALIZATION OF TWO GLYCOLYTIC DEHYDROGENASES IN WHITE SKELETAL MUSCLE

1966 ◽  
Vol 29 (1) ◽  
pp. 113-128 ◽  
Author(s):  
H. Dariush Fahimi ◽  
Morris J. Karnovsky
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Electron Flow ◽  
Aqueous Media ◽  
Cytochemical Localization ◽  
Diaphorase Activity ◽  
Adductor Magnus ◽  
Glycolytic Activity ◽  
Final Electron ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Soluble Enzymes

The cytochemical localization, by conventional methods, of lactate and glyceraldehyde-3-phosphate dehydrogenases is limited, firstly, by the solubility of these enzymes in aqueous media and, secondly, by the dependence of the final electron flow from reduced nicotinamide-adenine dinucleotide (NADH) to the tetrazolium on tissue diaphorase activity: localization is therefore that of the diaphorase, which in rabbit adductor magnus is mitochondrial. NADH has been found to have great affinity to bind in the sarcoplasmic reticulum, and, therefore, if it is generated freely in the incubation media containing 2,2',5,5'-tetra-p-nitrophenyl-3,3'-(3,3'-dimethoxy-4,4'-phenylene)-ditetrazolium chloride (TNBT) and N-methyl phenazonium methyl sulfate (PMS), it can bind there and cause a false staining. Since such a production of NADH can readily occur in the incubation media for glycolytic dehydrogenases due to diffusion of these soluble enzymes from tissue sections, the prevention of enzyme solubilization is extremely important. Fixation in formaldehyde prevented such enzyme diffusion, while at the same time sufficient activity persisted to allow for adequate staining. The incubation media contained PMS, so that the staining system was largely independent of tissue diaphorase activity. Application of these methods to adductor magnus of rabbit revealed by light microscopy, for both enzymes, a fine network which was shown by electron microscopy to represent staining of the sarcoplasmic reticulum. Mitochondria also reacted. These findings add further support for the notion that the sarcoplasmic reticulum is probably involved in glycolytic activity.

scholarly journals SOLUBLE ENZYMES OF NUCLEI ISOLATED IN SUCROSE AND NON-AQUEOUS MEDIA

1953 ◽  
Vol 37 (2) ◽  
pp. 177-187 ◽  
Author(s):  
Herbert Stern ◽  
A. E. Mirsky
Keyword(s):  
Adenosine Deaminase ◽  
Organic Solvents ◽  
Rat Liver ◽  
Nuclear Membrane ◽  
Aqueous Media ◽  
Isolated Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei ◽  
Soluble Enzymes ◽  
Soluble Components

Nuclei of calf thymus and liver and of rat liver were isolated in sucrose media and a number of their properties studied in relation to those of corresponding nuclei isolated in non-aqueous media with a view to determining their capacity to retain soluble components. The best preparations of sucrose nuclei were obtained from calf thymus. Cytochrome oxidase measurements and DNA/N ratios were far less sensitive than microscopic examination as indicators of purity when rat liver and calf thymus nuclei were compared. No satisfactory preparation of calf liver nuclei was obtained, contamination with whole cells having been appreciable; such preparations, nevertheless, could be used to advantage in the tests undertaken. DNA content of thymus nuclei isolated in sucrose was much the same as that of non-aqueous ones, pointing to a retention of soluble protein under aqueous conditions of isolation. That this net retention of protein was not due to the impermeability of the nuclear membrane was shown by the hydrolysis of the DNA upon addition of some crystalline DNAase to a sucrose suspension of nuclei. A comparative study of liver and thymus nuclei isolated in aqueous and non-aqueous media with respect to the soluble enzymes glucose-6-phosphate dehydrogenase, adenosine deaminase, and nucleoside phosphorylase yielded the following results: 1. Lyophilization of sucrose-isolated nuclei and their extraction with the organic solvents used in the non-aqueous procedure did not inactivate any of the enzymes tested. In the case of thymus the reverse was true, there being a marked increase in activity of all the enzymes studied. 2. In thymus, nucleoside phosphorylase and adenosine deaminase were active to approximately the same extent in nuclei isolated by either procedure. Glucose phosphate dehydrogenase alone was more active in sucrose-isolated nuclei, pointing to the possibility of an adsorption of this enzyme. 3. In rat liver nuclei isolated in sucrose, lyophilization and treatment with organic solvents revealed only the presence of some dehydrogenase. 4. The washing out of soluble enzymes was most markedly demonstrated in the case of calf liver. Only traces of the nucleoside enzymes were found in the sucrose-isolated nuclei, and in the case of the dehydrogenase only a half of that present in the non-aqueous nucleus remained. The main conclusions drawn were as follows:— 1. In sucrose media the nuclear membrane is ineffectual in preventing the inward or outward diffusion of protein. 2. The extent to which soluble proteins are retained by a nucleus isolated in sucrose appears to depend upon internal structural factors, such as the concentration of DNA in the nucleus. 3. With respect to determining the composition of nuclei in terms of soluble components, the sucrose isolation procedure is considered to be of indifferent merit and hence invalid for such a type of analysis.

scholarly journals Cytochemical localization of hydrogen peroxide production in the rat uterus.

1984 ◽  
Vol 32 (6) ◽  
pp. 674-676 ◽  
Author(s):  
Y Ishikawa ◽  
K Hirai ◽  
K Ogawa
Keyword(s):  
Plasma Membrane ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Surface Epithelium ◽  
Apical Plasma Membrane ◽  
Rat Uterus ◽  
Cytochemical Localization ◽  
Dense Deposits ◽  
Nadph Oxidation ◽  
Endometrial Epithelium ◽  
Reduced Nicotinamide Adenine Dinucleotide

A reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H)-dependent H2O2-generating activity of the rat uterus was investigated both electron cytochemically and biochemically. We tried to cytochemically demonstrate H2O2 generation from the oxidation of reduced NADH or NADPH using the cerium method. NADPH oxidation resulted in electron-dense deposits on the apical plasma membrane covering the microvilli of the surface epithelium of the lightly fixed endometrium. In control specimens incubated in a medium from which substrate was omitted, no such deposits were observed. The reduction of ferricytochrome c due to NADH oxidation was spectrophotometrically detected in the lightly fixed uterus. Absorption at 550 nm increased with the addition of NADH, but not with that of NAD. The reaction was weakened by preheating and adversely affected by the addition of superoxide dismutase, but it was not inhibited by adding 50 mM sodium azide. These results suggest that a kind of NAD(P)H oxidase, generating H2O2 via superoxide formation, may possibly be present on the apical plasma membrane of the rat endometrial epithelium.

scholarly journals Coupling of uridine-5'-diphosphate (UDP) formation and nicotinamide adenine dinucleotide (NAD+) reduction for cytochemical localization of glycosyltransferases.

1983 ◽  
Vol 31 (10) ◽  
pp. 1175-1182 ◽  
Author(s):  
G R Matyas ◽  
D J Morré
Keyword(s):  
Golgi Apparatus ◽  
Rat Liver ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine ◽  
Cytochemical Localization ◽  
Permeabilized Cells ◽  
Exogenous Glucose ◽  
Dense Deposits ◽  
Donor And Acceptor ◽  
Reduced Nicotinamide Adenine Dinucleotide

A technique applicable to the cytochemical localization of glycosyltransferases through a series of coupled enzyme reactions is described. Uridine-5'-diphosphate (UDP) formed by glycosyltransferases is first phosphorylated to uridine-5'-triphosphate (UTP) by nucleoside 5'-diphosphate kinase. The UTP plus exogenous glucose-1-phosphate is converted into UDP-glucose by uridine-5'-diphosphoglucose pyrophosphorylase. UDP-glucose is then oxidized by uridine-5'-diphosphoglucose dehydrogenase to form UDP-glucuronic acid and reduced nicotinamide adenine dinucleotide (NADH). The NADH is utilized by membrane-located NADH-ferricyanide oxidoreductases in the presence of a copper salt to form electron-dense deposits of cupric ferrocyanide (Hatchett's brown). Using this technique, galactosyltransferase has been localized in cisternae (including the central midregions of the cisternae) of Golgi apparatus isolated from rat liver. Reactivity is absent from the cis-most cisternae and membrane elements. The reaction is dependent on UDP-galactose and inhibited by ethylene diaminetetraacetic acid and puromycin. the latter is a known inhibitor of galactosyltransferase of rat liver Golgi apparatus. The reaction is adaptable by varying the sugar nucleotide donor and acceptor to any glycosyltransferase utilizing UDP-sugars (except UDP-glucose). Presently it is restricted to isolated membrane fractions and permeabilized cells due to the need for accessibility of reagents and coupling enzymes.

scholarly journals CYTOCHEMICAL LOCALIZATION OF CHOLINESTERASE ACTIVITY IN ADULT RABBIT HEART

1971 ◽  
Vol 19 (6) ◽  
pp. 376-381 ◽  
Author(s):  
MARTIN HAGOPIAN ◽  
VIRGINIA M. TENNYSON
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Papillary Muscle ◽  
Cholinesterase Activity ◽  
Acetylcholinesterase Activity ◽  
Rabbit Heart ◽  
Adult Rabbit ◽  
Cytochemical Localization ◽  
Excitation Contraction Coupling ◽  
Contraction Coupling ◽  
T System

The papillary muscle of the adult rabbit heart was studied by a modification of the Koelle-Friedenwald copper thiocholine technique for the localization of cholinesterase activity. Butyrylcholinesterase (BuChE), identified by its substrate and inhibitor specificity, is found mainly in the terminal sacs of the sarcoplasmic reticulum adjacent to the T system. The localization of the reaction product in this particular site suggests that BuChE may play a role in excitation-contraction coupling in the adult rabbit heart. The present findings are also discussed in comparison with our previous work on the localization of acetylcholinesterase activity in the embryonic rabbit heart.

scholarly journals The Cytochemical Localization of Oxidative Enzymes

1958 ◽  
Vol 4 (6) ◽  
pp. 747-752 ◽  
Author(s):  
D. G. Scarpelli ◽  
R. Hess ◽  
A. G. E. Pearse
Keyword(s):  
Cellular Localization ◽  
Oxidative Enzymes ◽  
Tetrazolium Salt ◽  
Direct Transfer ◽  
Metal Chelation ◽  
Cytochemical Localization ◽  
Black Metal ◽  
Respiratory Inhibitors ◽  
Diaphorase Activity ◽  
Tetrazolium Reduction

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.

scholarly journals Cytochemical localization of NADH oxidase in Candida albicans.

1977 ◽  
Vol 25 (3) ◽  
pp. 193-199 ◽  
Author(s):  
M Borgers ◽  
S De Nollin ◽  
F Thoné ◽  
H Van Belle
Keyword(s):  
Candida Albicans ◽  
Nicotinamide Adenine Dinucleotide ◽  
Oxidase Activity ◽  
Nadh Oxidase ◽  
Lethal Effect ◽  
Nicotinamide Adenine ◽  
Strong Increase ◽  
Cytochemical Localization ◽  
Prolonged Incubation ◽  
Reduced Nicotinamide Adenine Dinucleotide

The application of a recently published technique to localize reduced nicotinamide adenine dinucleotide oxidase activity is described in glutaraldehyde-fixed Candida albicans. The reaction product appears as a finely granular precipitate on the mitochondrial cristae and on the central vacuolar membrane, and, if present, on the vacuolar contents. Fixation should be kept to a minimum and prolonged incubation times up to 2 hr are necessary to show these reactive sites. The reaction appears to be strongly substrate-dependent and not affected by cyanide. Exposure of C. albicans cells to the antimycotic miconazole resulted in a strong increase in reduced nicotinamide, adenine dinucleotide and oxidase activity. The hypothesis is put forward that this enzyme, together with peroxidative and catalatic enzymes, may be implicated in the mechanism by which miconazole exerts its lethal effect on C. albicans.

Sign in / Sign up

Export Citation Format

Share Document