Isolation of oligomycin-sensitive adenosine triphosphatase from beef heart mitochondria and analysis of its fine structure

1. An oligomycin -sensitive ATPase was isolated and partially purified from beef heart mitochondria. The specific activity of ATPase sensitive to oligomycin of the fraction was five to eight times that of aged mitochondrial or of DNP-induced mitochondrial ATPase assayed under the same condition. 2. Electron micrographs of the partially purified oligomycin- sensitive ATPase reveal a structure in which headpieces are regularly attached by way of stalks to a thread-like structure derived from a superficial portion of base pieces. 3. A high concentration of the structured material coincided with a high activity of oligomycin-sensitive ATPase. When the headpieces were detached from the structure, the ATPase became insensitive to oligomycin. 4. The fraction of oligomycin -sensitive ATPase was essentially free of membrane structure and was contaminated with a small amount of cytochromes b and Cl but no cyt. a. Cytochrome concentrations of the preparations were indifferent to the activity of oligomycin sensitive ATPase. It follows that ATPase does not require cytochromes or membrane structure for its oligomycin sensitivity. 5. From these results it seems that the factor rendering ATPase sensitive to oligomycin should be contained in the stalks and/or the thread-like portion of basepieces of the structure. The structure is the simplest unit of oligomycinsensitive ATPase as yet obtained. 6. The structure was called &#34;oligomycin-sensitive ATPase particles&#34; (abbreviated as OSA particles). A unit of OSA particles consists of a headpiece attached by a stalk to a portion of base piece.</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

Electron microscopic observation of mitochondrial DNA isolated from a human kidney: circular dimers in histologically normal organ mitochondria

Circular DNA isolated from human kidney mitochondria was studied by electron microscopy. I. Mean contour length of monomers of the mitochondrial DNA was 4.96 ± SE 0.28 /&#956; 2. The complex molecules (oligomers) of mitochondrial DNA were observed in frequency of 6.2 per cent. Among them circular dimers accounted for two per cent of all circular DNA molecules. 3. Circular DNA fibers with an intermediate perimeter between the&#12288;monomer and dimer, and with a contour length shorter than 3 &#956; were occasionally observed. 4. Some discussions were made on the emergence of the circular dimer.</p

Molecular basis of structure and function of the microvillus membrane of intestinal epithelial cells

Correlation of molecular structure with biochemical functions of the plasma membrane of the microvilli of intestinal epithelial cells has been investigated by biochemical and electron microscopic procedures. Repeating particles, measuring approximately 60 &#197;in diameter, were found on the surface of the microvilli membrane which had been isolated or purified from rabbit intestinal epithelial cells and negatively stained with phosphotungstic acid. These particles were proved to be inherent components of the microvillus membrane, attached to the outer surface of its trilaminar structure, and were designated as the elementary particles of the microvilli of intestinal epithelial cells. Biochemical and electron microscopic identification of these elementary particles has been carried out by isolation of the elementary particles with papain from the isolated microvillus membrane, followed by purification of the particles by chromatographies on DEAE-cellulose and Sephadex columns. The partially purified particles containing invertase and leucine aminopeptidase are similar in size and structure to those of the elementary particles in the microvillus membrane. Evidence indicates that each of the elementary particles coincide with or include an enzyme molecule such as disaccharidase or peptidase, which carry out the terminal hydrolytic digestion of carbohydrates and proteins, respectively, on the surface of the microvillus membrane. Magnesium ionactivated adenosine triphosphatase and alkaline phosphatase cannot be solubilized with papain but remains in the smooth-surface membrane after the elementary particles have been removed. Cytochemical electron microscopic observation revealed that the active site of magnesium ion-activated adenosine triphosphatase is localized predominantly in the inner surface of the trilaminar structure of the microvillus membrane.</p

Flavin and cytochrome contents in the mitochondria of the heart and liver

With a certain fixed methods of analyses, we carried out the determination of flavins and cytochromes in the mitochondria (Mt) and electron transfer particles (ETP) of the heart and liver of rats and cows, and made a comparison of the data with one another. Our findings may briefly be summarized as follows. 1. The concentration of each component of the beef heart mitochondria proved to be 0.47 for acid extractable flavins; 0.22 for acid nonextractable flavin; O. 75 for cytochrome (cyt.) a; 0.58 for cyt. b; and O. 51 for cyt. C + Cl, all units being m&#956; mole per mg of protein. 2. In the beef liver mitochondria it was 0.46 for acid extractable flavins; 0.18 for acid non-extractable flavin; 0.092 for cyt. a; 0.089 for cyt. b; and 0.122 for cyt. C+Cll likewise all units in term of m&#956; mole per mg of protein. 3. In the case of rat heart mitochondria, it was found to be O. 42 for acid extractable flavins; 0.22 for acid non-extractable flavin; 0.88 for cyt. a; 0.41 for cyt. b; and 0.62 for cyt. C + Cll all in m&#956; mole per mg of protein. 4. In the rat liver mitochondria it was 0.56 for acid extractable flavins; 0.19 for acid non-extractable flavin; 0.20 for cyt. a; 0.14 for cyt. b; and 0.19 for cyt. C+Cl. 5. The concentration ratios of Fs, cyt. a and cyt. b of the mitochondria, what are considered to be intrinsic and fixed components of the mitochondrion. to those of the electron transfer particles were 1. 3 in both the beef heart and the rat heart, while 2.2 in the beef liver and 2.1 in the rat liver. 6. These findings were compared with the data reported by other workers, and also a discussion was made on the molecular organization of the mitochondrial inner membrane.</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

Purification and fine structure of reduced coenzyme Q-cytochrome C reductase in the mitochondrial membrane

For the purpose of revealing the molecular organization of the mitochondrial membrane the authors attempted to clarify the fine structure of reduced coenzyme Q-cytochrome c reductase and also studied how the CoQH2-cyt. c reductase is arranged in the mitochondrial membrane by systematic analyses of fractions from the purification process of CoQH2-cyt. c reductase. 1. Purified CoQH2-cyt. c reductase contained high concentration of cyt. b (9.5 m&#956;moles per mg protein) and cyt. Cl (4.5 m&#956;moles per mg protein), and was almost free from cyt. c, a, flavoproteins, primary dehydrogenases and ATPase. The enzyme complex also showed a high specific activity (48 &#956;moles of cyt. c reduced per mg protein per min at 30°). 2. CoQH2-cyt. c reductase was composed of particles of about 120 &#197; in diameter with irregular form, some time exhibiting electron opaque cores. In the loose aggregates of the particles, the size of each particle was about 95 &#197; in diameter. 3. An intimate correlation was observed between the particles of CoQH2cyt. c reductase and those on the surface of the NADH-cyt. c reductase fraction. 4. Regular arrays of uniform particles (about 82 &#197; in diameter with a center to center distance of about 100 &#197;) were observed on the surface of the submitochondrial membrane (brown membrane) obtained from beef heart mitochondria by treatment with deoxycholate (0.1 mg / mg protein) and KCl (72 g/l). The correlation between these particles and CoQH2-cyt. c reductase was discussed.</p

Studies on bleomycin-induced repair DNA synthesis in permeable mouse ascites sarcoma cells.

To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He) cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta) and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s).</p

Uptake of nicotinamide adenine dinucleotide and excretion of its degradation pro­ducts by tissue culture cells

For the purpose to reveal the mechanism of uptake and degradation of NAD by cells, the authors conducted the observation on the L cells cultured in the medium containing NAD and the following results have been obtained. 1. NAD in the medium is taken up by the cells in its intact form, reaching about twice the value of the control. 2. The spontaneously degraded products of NAD, nicotinamide and adenine dinucleotide ribose, in the same molar concentration as NAD used in the present experiment, have no effect on the NAD content of L cells. 3. The NAD taken up by the cells is degraded into nicotinamide mononucleotide (NMN) and adenine mononucleotide (AMP) by pyrophosphatase including NADpptase and excreted in the medium. Unexpectedly the ingested NAD is not degraded by NADase in the L cell. 4. L cells metabolize the same amount of NAD as that contained originally in the cell for about ten minutes, as calculated from the amount of NMN excreted in the medium.</p

Properties of ATP-ase of the microvillus membrane isolated from epithelial cells of rabbit small intestine

For the purpose to investigate the physiological functions of microvillus ATPase, general properties of the enzyme were studied on the microvillus membranes isolated from rabbit intestinal epithelial cells. 1) ATPase of the microvillus membranes was activated with Mg2+. Mg.ATP complex was thought to be a subStrate of the enzyme. The Michaelis constant for ATP of the ATPase was a value of 0.8 to I .0 mM. 2) The microvillus ATPase was also activated with Ca2+, but the affinity was lower than a half of that of Mg2+. 3) The optimum pH of the ATPase was about 7.8. 4) Activity of the microvillus ATPase was markedly inhibited by treating with deoxycholate (DOC), and the activity inhibited was partially restored by washing the microvillus membrane with distilled water. The structure of the membranes destroyed by treating with DOC was also partially restored by the same procedure. 5) Ultrasonic treatment also markedly destroyed the microvillus membrane and inhibited ATPase activity. Damaged ultrastructure and ATPase activity both were partially restored by treating with phospholipid, EPL. 6) Simultaneous presence of Na+ and K + stimulated scarcely the ATPase of purified microvillus membranes. 7) The microvillus ATPase was slightly activated in the presence of n-glucose. Phloridin gave little effect on the activity of the microvillus ATPase.</p