An analytical study on the reduction of neotetrazolium chloride by the terminal electron transport system

In order to determine the steps with which the reaction of neotetrazolium chloride reduction conjugates in the terminal electron transport system, an analytical study on the neotetrazolium reduction by tissue homogenates was carried out using various substrates such as sodium succinate, p-phenylenediamine, sodium malate, sodium &#945;-glutamate and DPN, and inhibitors such as sodium malonate, potassium cyanide and antimycin A, as the results the following conclusions were drawn. 1. The reaction of neotetrazolium reduction by tissue homogenate using sodium succinate as substrate is mainly the succinoxidase system reaction; and the reaction takes place conjugating about 50 per cent in the step of the succinic dehydrogenase system (succinic dehydrogease, cytochrome b and cytochrome C1), of these about 15 per cent conjugates in the step prior to the antimycin A sensitive step and 35 per cent in the step itself; and about 50 per cent in the step of cytochrome c oxidase. 2. In the case using p-phenylenediamine as substrate the reaction of neotetrazolium reduction is the reaction due to the activity of cytochrome c-cytochrome oxidase system; and when p-phenylenediamine is used with the sufficient amount of cytochrome c, the reaction appears to be dependent on cytochrome c oxidase activity. Neotetrazolium reduction in all these reactions takes place conjugating in the step of cytochrome c oxidase. 3. In the case where DPN and substrates taking DPN as a coenzyme are used, the reaction of neotetrazolium reduction is mainly the reaction conjugating at the step below antimycin A sensitive step in the DPNHcytochrome c reductase system (flavoprotein, cytochrome b and cytochrome c;), probably with the flavoprotein of DPNH-dehydrogenase. 4. Endogenous dehydrogenase reactions are the sum total reactions conjugating at the steps prior to the antimycin A sensitive step in the terminal electron transport system and with other various reduction systems which are not inhibited by antimycin A.</p

Cytochemicar demonstra­tion of the sites of activity of the terminal electron transport system with the electron microscope

In an attempt to pursue the relationship of the fine structure of a cell to the biochemical function, the author at first tried to demonstrate cytochemically the actual sites of activity of enzymes in the terminal electron transport system involved in energy production with the use of the electron microscope. Namely, cytochemical reactions were performed by using potassium tellurite, a heavy metal salt, and then the author succeeded in the electron microscopic detection of the enzymes by freezing-drying method and by means of formalin fixation, strong reducing agents and osmium tetroxide fixation. As the results the author has been able to verify that the reactions of the enzymes belonging to the terminal electron transport system are found localizing in the mitochondria being arranged fairly densely and continuously on the critae and partially on the membrane, although some differences in the grade of the activity are found in each mitochondria even in one cell and a marked difference between the mitocndria belonging to the different kinds of cells. Furthermore, it has been clarified that the activity of the endogenous dehydrogenase system (mainly DPNH- or TPNH-dehydrogenase and others) is chiefly strong in cristae, and that the succinoxidase system exists both in cristae and membrane.</p

New methods for the histochemical and cytochemical demonstrtion of cytochrome c oxidase and of cytochrome c- cytochrome oxidase system

New histochemical and cytochemical methods for the demonstration of cytochrome c oxidase and of cytochrome c-cytochrome oxidase system are described, using neotetrazolium chloride in the presence of p phenylenediamine with or without additional cytochrome c. These enable the cytochemical visualization of the sites of enzyme activity at the intracellular level in fresh cell suspensions and in fresh tissue blocks under aerobic conditions, and permit the histochemical visualization of the distribution of the enzyme activity in various tissues in frozen tissue sections. The colorimetric estimation of the enzyme activity is also possible in the combination of the methods previously described.</p

New colorimetic methods for the estimation of cytochrome c oxidase and of cytochrome c-cyto-chrome oxidase system

New colorimetric methods for the estimation of the activities of cytochrome c oxidase and of cytochrome c-cytochrome oxidase system in tissue homogenates are described, using neotetrazolium chloride in the presence of p-phenylenediamine with or without additional cytochrome c. Optimal time of incubation, optimal concentration of the incubation medium and amounts of tissue, and simple method for the extraction of the reduced neotetrazolium chloride were determined. The reduction of neotetrazolium chloride was proportional to the amount of enzyme. Using this method, colorimetric estimations of cytochrome c-cytochrome oxidase system activity in the kidney, heart, liver, brains, and skeletal muscle were made. The procedures of these methods are very simple, and they are considered to be feasible in the combination with histochemical demonstrations of these enzyme activities.</p

Histo-chemical and cytochemical studies on the succinic dehydroge-nase system with three ditetrazolium salts, NT, Nitro-NT, and Nitro-Bt

1. Histochemical and cytochemical studies with respect to the sites of reaction were made on the succinic dehydrogenase system activity of human and animal tissues using ditetrazolium salts, namely, neotetrazolium chloride, nitro-neotetrazolium chloride, and nitra-blue tetrazolium chloride. 2. The advantages and disadvantages of each ditetrazolium salt for histochemical and cytochemical purposes and the reaction taking place in frozen tissue sections and that in fresh tissue blocks were compared, and the method of procedure suitable for each condition was established with some modification. 3. Selecting conditions suitable for cytochemical purpose, it was shown that the reaction took place at the sites coinciding with mitochondria, and the distribution of the enzyme reaction was also examined. In addition, several new findings in the brains and other tissues cytochemically made clear were pointed out.</p

Analytical Studies on the Colorimetric Estimation of the Activity of Succinic Dehydrogenase System with Neotetrazolium Chloride

Despite the excellence in bringing about reactions, there are still many difficulties and discrepancies in the use of neotetrazolium chloride for measuring the activity of succinic dehydrogenase system of tissue homogenate. Therefore, with the purpose to find out a measuring method simpler and more accurate, the authors performed some experimets, first on the enzymic reactions of tissue homogenate and then after stopping these reactions by using various agents, tested the ease with which reduction products are extracted with various solvents, and finally compared the absorption spectra of these liquid extracts with those of the products reduced chemically by sodium hydrosulfite. As the results, the extraction of the reduction products with acetone-ether (mixed in equal proprtion v/v) after stopping the reaction with 10 per cent formalin solution was found to be the simplest and best method, and moreover, the absorption spectra of extracts of the products obtained by enzymatical or chemical reduction proved to he uniform and the both of which showed the maximum absorption at the wave length of 520 mμ. In addition, the concentrations of sodium succinate and neotetrazolium chloride, the content and kinds of tissues, pH values and concentration of phosphate buffer solutions, the duration of reaction, aerobic or anaerobic conditions, various factors either enhancing or in hibitory to reaction, were all regulated absolutely or relatively; and by drawing absorption curves of the reduction products, these enzymatic reactions were analyzed and scrutinyzed Then the authors established a method which is general, simple and accurate in measuring the enzymatic activities of a small quantity of fresh tissues in contrast with those of hist chemically stained specimens

An electron-microscopic study on lipogenesis

With the purpose to elucidate morphologically the site where fat synthesis takes place in the cell, electron-microscopic observation has been conducted on the interscapular brown fat tissue of mice at various periods of carbohydrate introduction after starvation. By starving mice, the depot lipids in the brown fat have been discharged almost completely, and the carbohydrate introduction has caused the biosynthesis of lipids from carbohydrtates in the same tissue. Observations on the tissues proved that the lipogenesis in the brown fat tissue cells takes place in the ground substance keeping the intimate correlation with the endoplasmic reticulum but not in the mitochondria.</p