scholarly journals The ultrastructural cytochemistry of lactic dehydrogenase, succinic dehydrogenase, dihydro-nicotinamide adenine dinucleotide diaphorase and cytochrome oxidase activities in hair cell mitochondria of the guinea pig cochlea.

1975 ◽  
Vol 23 (3) ◽  
pp. 216-234 ◽  
Author(s):  
G J Spector
Keyword(s):  
Hair Cell ◽  
Cytochrome Oxidase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Succinic Dehydrogenase ◽  
Lactic Dehydrogenase ◽  
Nicotinamide Adenine ◽  
Fixation Method ◽  
Inner Mitochondrial Membrane ◽  
Tetrazolium Chloride ◽  
Outer Compartment

The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.

scholarly journals NONDROPLET ULTRASTRUCTURAL DEMONSTRATION OF CYTOCHROME OXIDASE ACTIVITY WITH A POLYMERIZING OSMIOPHILIC REAGENT, DIAMINOBENZIDINE (DAB)

1968 ◽  
Vol 38 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Arnold M. Seligman ◽  
Morris J. Karnovsky ◽  
Hannah L. Wasserkrug ◽  
Jacob S. Hanker
Keyword(s):  
Cytochrome Oxidase ◽  
Respiratory Chain ◽  
Oxidase Activity ◽  
Oxidative Polymerization ◽  
Succinic Dehydrogenase ◽  
Inner Mitochondrial Membrane ◽  
Electron Microscopic ◽  
Cytochrome Oxidase Activity ◽  
Outer Compartment ◽  
Electron Microscopes

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.

scholarly journals MORPHOLOGICAL AND CYTOCHEMICAL PROPERTIES OF THE HOLOCRINE CELLS IN THE EPIDIDYMIS OF THE MOUSE

1964 ◽  
Vol 12 (8) ◽  
pp. 628-639 ◽  
Author(s):  
JAN MARTAN ◽  
JOHN M. ALLEN
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Periodic Acid ◽  
Succinic Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Central Nucleus ◽  
Nicotinamide Adenine ◽  
Secretory Cells ◽  
Basal Cells ◽  
Periodic Acid Schiff ◽  
Highly Active

Holocrine secretory cells have been identified in the epithelium of the epididymal canal of the mouse. These cells develop from basal cells. During their differentiation they grow toward the lumen of the epididymal canal and come to form club-shaped structures with an expanded apical portion, a central nucleus and a thin stalk-like connection to the basement membrane. Mature holocrine cells are characterized by their high acid phosphatase and aliesterase activity. They also are highly active for succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase, and nicotinamide adenine dinucleotide phosphate diaphorase. Nucleoside diphosphatase, thiamine pyrophosphatase, adenosine triphosphatase, and alkaline nucleoside phosphatase are also found in these cells. These cells are also characterized by their reactivity with the Aoyama and periodic acid-Schiff reactions. They react moderately with the molybdate and Luxol Fast Blue MBS reactions for choline containing compounds. Mature holocrine cells may disintegrate in situ or may be discharged in toto into the lumen of the epididymal canal. Glycerylphosphorylcholine was identified in extracts prepared from sperm-free epididymides of mice. Glycerylphosphorylcholine reacts with Aoyama and periodic acid-Schiff reactions as do mature holocrine cells. This fact coupled with the identification of choline containing material in holocrine cells suggests that they may be one site for the formation of glycerylphosphorylcholine.

FACTORS INFLUENCING TETRAZOLE REDUCTION BY REDUCED NICOTINAMIDE–ADENINE DINUCLEOTIDE IN PIGEON-HEART MITOCHONDRIA

1967 ◽  
Vol 45 (2) ◽  
pp. 299-307 ◽  
Author(s):  
C. L. Talesara ◽  
M. C. Blanchaer
Keyword(s):  
Adenosine Triphosphate ◽  
Respiratory Chain ◽  
Nicotinamide Adenine Dinucleotide ◽  
Inorganic Phosphate ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Heart Mitochondria ◽  
Nicotinamide Adenine ◽  
Tetrazolium Chloride ◽  
Reduced Nicotinamide Adenine Dinucleotide

The effect of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate and inorganic phosphate on the reduction of 2-(p-iodophenyi)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT) to its formazan by reduced nicotinamide-adenine dinucleotide (NADH) was studied in pigeon-heart mitochondria. Formazan production was followed at 540 mμ in 2.2 ml medium containing 0.4–0.5 mg mitochondrial protein, 0.22 M mannitol, 0.067 M sucrose, 0.02 M Tris–chloride, 0.02 mM EDTA, 0.5–3.0 mM INT, and 38 μM NADH at pH 7.2 and 28 °C. By means of the respiratory inhibitors Amytal, rotenone, antimycin A, and cyanide, it was shown that INT diverts electrons from the respiratory chain principally at the flavoprotein level. In contrast to its inhibitory effect on "the O2-linked oxidation of NADH, 10 mM adenosine triphosphate stimulated the reaction rate and formazan yield in the present system. Equimolar inorganic phosphate also increased the initial velocity but adenosine diphosphate and adenosine monophosphate did not. Preliminary kinetic studies suggest that NADH, but not INT, combines with the form of NADH dehydrogenase in the respiratory chain with which adenosine triphosphate reacts.

scholarly journals Lack of Maternal Glutamate Cysteine Ligase Modifier Subunit (Gclm) Decreases Oocyte Glutathione Concentrations and Disrupts Preimplantation Development in Mice

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2806-2815 ◽  
Author(s):  
Brooke N. Nakamura ◽  
Thomas J. Fielder ◽  
Yvonne D. Hoang ◽  
Jinhwan Lim ◽  
Lisa A. McConnachie ◽  
...  
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Blastocyst Stage ◽  
Preimplantation Development ◽  
Nicotinamide Adenine ◽  
Lower Percentage ◽  
Inner Mitochondrial Membrane ◽  
Cell Stage ◽  
Glutamate Cysteine Ligase ◽  
Reduced Nicotinamide Adenine Dinucleotide

Glutathione (GSH) is the most abundant intracellular thiol and an important regulator of cellular redox status. Mice that lack the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH synthesis. Nicotinamide nucleotide transhydrogenase, an inner mitochondrial membrane protein, catalyzes the interconversion of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate; reduced nicotinamide adenine dinucleotide phosphate is required for reduction of GSH disulfide. Previous work supports roles for GSH in preimplantation development. We hypothesized that Gclm−/− mice have increased preimplantation embryonic mortality and that this effect is enhanced by absence of a functioning Nnt gene. Gclm−/− females produced significantly fewer pups per litter than Gclm+/+ littermates. Numbers of oocytes ovulated in a natural estrous cycle or upon superovulation did not differ by genotype. Fewer uterine implantation sites were observed in the Gclm−/− females. Prepubertal Gclm−/− and Gclm+/+ females were superovulated, then mated overnight with a Gclm+/+ male. At 0.5 d postcoitum, Gclm−/− females had significantly lower percentages of zygotes with two pronuclei and higher percentages of zygotes with one pronucleus than Gclm+/+ or Gclm+/− females. At 3.5 d postcoitum, a significantly lower percentage of blastocyst stage embryos was recovered from uteri of Gclm−/− females than Gclm+/+ females. Embryonic development to the blastocyst stage, but not the two-cell stage, was significantly decreased after in vitro fertilization of oocytes from Gclm−/− females compared with Gclm+/+ females. The Nnt mutation did not enhance the effects of Gclm genotype on female fertility. These results demonstrate critical roles for maternal GSH in supporting normal preimplantation development.

ENZYME INHIBITION BY DERIVATIVES OF PHENOTHIAZINE: III. CATALASE, CYTOCHROME OXIDASE, AND DEHYDROGENASES

1942 ◽  
Vol 20b (12) ◽  
pp. 284-290 ◽  
Author(s):  
H. Bruce Collier ◽  
Della E. Allen
Keyword(s):  
Cytochrome Oxidase ◽  
Succinic Dehydrogenase ◽  
Lactic Dehydrogenase ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Leuco Form ◽  
Dehydrogenase System ◽  
Living Organisms ◽  
Relationship Of ◽  
Derivatives Of

The inhibition of liver catalase and of cytochrome oxidase by leucophenothiazone has been confirmed by manometric methods. Phenothiazine sulphoxide is a powerful catalase inhibitor, its activity being much greater at pH 5.3 than at neutrality.The succinoxidase activity of beef heart is inhibited by phenothiazone and by thionol. Phenothiazone in its oxidized form acts upon the succinic dehydrogenase, while the leuco form inhibits the cytochrome oxidase.Phenothiazone is reduced by the yeast lactic dehydrogenase system, and the enzyme activity is markedly decreased. Urease is also partially inhibited, whereas the effect of phenothiazone upon d-amino-acid oxidase is almost negligible.The possible relationship of these findings to the action of phenothiazine upon living organisms is discussed.

A Continuous Spectrophotometric Method for the Determination of Leucine Aminopeptidase

1968 ◽  
Vol 14 (6) ◽  
pp. 555-564 ◽  
Author(s):  
J A Knight ◽  
D T Hunter
Keyword(s):  
Spectrophotometric Method ◽  
Nicotinamide Adenine Dinucleotide ◽  
Leucine Aminopeptidase ◽  
Lactic Dehydrogenase ◽  
New Method ◽  
Nicotinamide Adenine ◽  
Glutamic Pyruvic Transaminase ◽  
Peptide Hydrolase ◽  
Reduced Nicotinamide Adenine Dinucleotide

Abstract A new method to assay the enzyme, leucine aminopeptidase (LAP; L-leucyl-peptide hydrolase—3.4.1.1), is described. The technic is an extension of the conventional glutamic-pyruvic transaminase (GPT) reaction. Serum LAP is allowed to react with the substrate L-leucyl-L-alanine to quantitatively generate alanine. This alanine, in the presence of excess α-ketoglutaric acid and GPT, rapidly forms pyruvate, which is converted to lactate by excess lactic dehydrogenase (LDH). Reduced nicotinamide adenine dinucleotide (NAD) is quantitatively oxidized in this reaction. The change in reduced NAD concentration is followed by continuous spectrophotometry at 340 mµ.

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