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Most synapses in the central nervous system exhibit a prominent electron-opaque specialization of the postsynaptic plasma membrane called the postsynaptic density (PSD). We have developed a procedure for the isolation of PSDs which is based on their buoyant density and their insolubility in N-lauroyl sarcosinate. Treatment of synaptic membranes with this detergent solubilizes most plasma membranes and detaches PSDs from the plasma membrane so that they can be purified on a density gradient. Isolated PSDs appear structurally intact and exhibit those properties which characterize them in tissue. The isolated PSDs are of the size, shape, and electron opacity of those seen in tissue; they stain with both ethanolic phosphotungstic acid and bismuth iodide-uranyl lead and the fraction contains cyclic 3',5'-phosphodiesterase activity. Quantitative electron microscope analysis of the PSD fraction gives an estimated purity of better than 85%. Inasmuch as the PSD is associated primarily with dendritic excitatory synapses, our PSD fraction represents the distinctive plasma membrane specialization of this specific synaptic type in isolation.

Experimental data are presented on the intracellular localization in rat liver of three enzymes which are involved in the biosynthesis of phosphatidylethanolamine and diphosphatidylglycerol. These enzymes are phosphatidylserine decarboxylase, CDP-diglyceride-l-alpha-glycerophosphate phosphatidyl transferase, and phosphatidylethanolamine-l-serine phosphatidyl transferase. It was found that the first two enzymes are primarily mitochondrial while the latter enzyme is primarily microsomal. The intracellular sites for the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol are discussed, and the implications of their sites of biosynthesis on the assembly processes involved in the biogenesis of mitochondria are considered.

Hepatic parenchymal cells from adult rats, established in vitro as a monolayer, have been evaluated by electron microscopy. Within 24 h after the initial seeding, the incubated cells were polygonal and in close apposition with three to six neighboring cells. The ultrastructure of the monolayer cells was examined at this time and after 3 and 10 days of incubation. With the exception of a few enlarged mitochondria, organelles in both the 1- and 3-day monolayer cells were indistinguishable quantitatively and morphologically from those found in the intact liver. After 10 days of incubation, however, the rough-surfaced endoplasmic reticulum (RER) had become dilated and vesiculated. In all cells studied, portions of RER were found in a close spatial relationship to mitochondria. From its frequency, this association appeared to be more than fortuitous, and the organelle complex may represent a functional unit necessary for new membrane formation, as suggested previously. The Golgi complexes of 1- and 3-day cells contained very low density lipoprotein-sized particles, which suggests that the monolayer cells synthesize lipoproteins. These electron microscope observations demonstrate that adult hepatic parenchymal cells in monolayer retain for several days the subcellular structural elements characteristic of normally functioning hepatocytes.

Laser irradiated mitochondria in rat myocardial cells were analysed cytochemically for succinic dehydrogenase and with the electron microscope for ultrastructure. The least severe lesion type did not alter the SDH activity of the mitochondria. The major ultrastructural alteration observed was an electron-dense region corresponding to a phase-dark spot in the light micrographs. This lesion appeared to be restricted in depth and width within the irradiated structure. The damage was primarily localized to the intercristae matrix. The cristae appeared normal. © 1973.

The axonal migration of nucleic acids and their precursors has been studied following injection of radioactive uridine or thymidine into a single eye of newhatched chicks. After monocular injection of tritiated uridine, a progressive increase was observed in the specific activity of RNA of the optic lobe contralateral to the injected eye. This accumulation was first apparent 18 hours after injection and increased for at least 6 days. There was also a lesser accumulation of trichloracetic acid soluble radioactivity in this contralateral lobe relative to the ipsilateral lobe. RNA was prepared from morphological fractions of optic lobes after differential centrifugation. Radioactive analysis of these RNA fractions suggested that mitochondrial, ribosomal and transfer RNA all migrated axonally to a considerable degree. At neither 4 hours or 4 days after monocular injection of tritiated thymidine, was there any excess of radioactivity in DNA of lobes contralateral to the injected eye relative to DNA of ipsilateral lobes. Thus there was no evidence for the flow of DNA along the axon. The data suggest that a wide variety of RNA species synthesised in the nerve cell body migrate distally and appear at nerve endings. © 1971 Springer-Verlag.

Leucocyte migration tests with rat liver mitochondria have been performed in eighteen patients with juvenile diabetes mellitus and in fifteen healthy controls. Leucocytes from normal subjects showed no inhibition of leucocyte migration. whereas inhibition occurred in twelve out of eighteen diabetics. The active component appeared to be in the inner membranes of the mitochondria in seventeen patients, whilst leucocytes from one patient showed inhibition to outer membrane fragments. Leucocyte migrations tests were performed in a further thirty-five patients, again using rat liver mitochondria as antigen, and sera from these patients was investigated for the presence of tissue autoantibodies. In those patients having a positive leucocyte migration test there was a higher incidence of circulating antibodies than in the group where white cells showed no inhibition but this did not reach statistical significance. [ABSTRACT FROM AUTHOR]

1. In the presence of succinate as an oxidation substrate, neurotoxins α, β and γ induce the following. Firstly, an increasing stimulation of oxygen uptake, which is potentiated by 25 µm Ca2+. Mg2+ 1.3 mM completely inhibits the effect of toxin α but not of toxins β and γ. Secondly, a depletion of the Mg2+, Ca2+ and K+ content of the water-soluble and membrane-bound mitochondrial compartments, following complex kinetics, which suggest a multistep interaction mechanism of the toxins with the mitochondria. 25 µM Ca2+ also potentiates the effect of the toxins on these ionic flows. Thirdly, no decrease of turbidity with toxin α, and a limited decrease with toxins β and γ. 2. In the absence of respiration, the neurotoxins induce a cationic depletion, the kinetics of which are different than with succinate, suggesting an instantaneous maximal effect on the inner membrane. Toxins β and γ (but not α) induce, under these conditions, a turbidity decrease of large amplitude, which is proportional to the amount of toxin added and tends to reach a maximum. With γ toxin this turbidity decrease is faster than the rate of water uptake (which never exceeds 18%) indicating that it is due rather to structural modifications than to swelling. The same is observed with β toxin, provided the mitochondrial protein concentration to be lower than 0.7 mg/ml. For higher concentrations, a continuous decrease of turbidity together with a considerable uptake of water probably reflects the onset of phospholipasic activities. 3. It is postulated that structural modifications of the mitochondrial membranes are initiated which lead to the loss of their selective impermeability. The simultaneous loss of respiratory control with succinate may be due to the direct (though Ca2+-potentiated) displacement of the fraction of the membrane-bound Mg2+ ions which controls its energy-transducing properties. 4. In addition, correlations between the effects of the toxins on mitochondria and their neurotoxicity in vivo are discussed. [ABSTRACT FROM AUTHOR]

Arginases have been found to be located on the external side of the inner mitochondrial membrane of chicken kidney and liver. Transamidinase has been detected within the liver mitochondrial matrix space. Arrases and transamidinase act upon two different intracellular arginine pools. Penetration of arginine into the matrix space occurs only in respiring mitochondria and in the presence of anions such as acetate and phosphate; D-arginine, L-ornithine, D-ornithine and L-lysine penetrate with the same modalities. L-Histidine penetrates only kidney mitochondria. Because of transamidinase compartmentation, the rate of creatine synthesis is influenced by the rate of penetration of arginine into the mitochondria. [ABSTRACT FROM AUTHOR]

(Adenine-14C) or (γ-32P)-labelled 2,2'[1-(9-adenyl)-1'-(tri-, diphosphoryl-oxymethyl)]-dihydroxydiethylether (rroANP) was obtained from AArp by cleavage of the C-2'-C-3' bond by sodium periodate oxidation and subsequent borohydride reduction. Binding of rroANP to rat liver mitochondria revealed carrier-linked (atractyloside-sensitive) and unspecific (atractyloside-insensitive) binding but no transfer across the inner mitochondrial membrane. Kinetic data indicate rroANP as a competitive inhibitor for ANP uptake with Ki = 9.3 × 10-5 M. Experimental rroANP confirmed that an intact adenine base and three anionic charges of the phosphate chain are essential for the recognition between ANP-carrier and nucleotide but insufficient for the induction of a transmembrane ANP exchange. In addition mobilisation of the carrier-nucleotide complex requires an intact ribofuranoside ring system. [ABSTRACT FROM AUTHOR]

A cytochrome c derivative from which iron is removed has been prepared and characterized. Several lines of evidence indicate that native and porphyrin cytochrome c have similar conformations: they have similar elution characteristics on Sephadex gel chromatography; in both proteins the tryptophan fluorescence is quenched and the pK values of protonation of the porphyrin are identical. Porphyrin cytochrome c does not substitute for native cytochrome c in either the oxidase reaction or in restoring electron transport in cytochrome-c-depleted mitochondria. It does however competitively inhibit native cytochrome c in these reactions, the Ki for inhibition being larger than the Km for reaction. The absorption and emission spectra, and the polarized excitation spectrum of the porphyrin cytochrome c are characteristic of free base porphyrin. The absence of fluorescence quenching of porphyrin cytochrome c when the protein is bound to cytochrome oxidase suggests that heme to heme distance between these proteins is larger than 0.5 to 0.9 nm depending upon orientation. Binding of the porphyrin cytochrome c to phospholipids or to mitochondria increases the fluorescence polarization of a positively polarized absorption band, which indicates that the bound form of the protein does not rotate freely within the time scale of relaxation from the excited state. [ABSTRACT FROM AUTHOR]

The ratio of the specific radioactivities of 3-hydroxybutyrate: citrate was determined in rat liver mitochondria which were incubated in the presence of [1-14C]palmitate, pyruvate, bicarbonate, ATP, phosphate and malonate. Without compartmentation this ratio would maximally be 2, however, under our conditions values of 2.5-3.7 were observed. In further experiments with mitochondria, the sensitivity of pyruvate carboxylase for acetyl-CoA produced from various precursors was tested. It was found that acetyl-CoA produced from L-acetylcarnitine or by oxidation from either pyruvate, octanoate or palmitylcarnitine but not from leucine led to a stimulation of pyruvate carboxylation. These results demonstrate a compartmentation of acetyl-CoA in liver mitochondria. The further finding that different mitochondrial fractions showed varying ratios of specific radioactivities of 3-hydroxybutyrate: citrate indicates that the observed compartmentation may be explained by the existence of different types of mitochondria with varying enzyme patterns and acetyl-CoA pools. [ABSTRACT FROM AUTHOR]

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13 000 x g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13 000 x g supernatant fraction. In neural CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm³, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly(A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA. [ABSTRACT FROM AUTHOR]

Microbodies, designated as peroxisomes because of their enzyme complement, have been isolated from methanol-grown cells of Candida boidinii. Spheroplast lysates were separated on non-continuous Ficoll density gradients, resulting in a mitochondrial fraction and a peroxisome fraction. Estimates of purity using the mitochondrial enzyme markers suggested that the contamination of mitochondria in the peroxisome fraction was about 2-3 %. As shown by electron microscopy the peroxisomes were 0.4-0.6 jim in diameter and contained crystalloid inclusions. Alcohol oxidase and catalase, which catalyse the oxidation of methanol to formaldehyde in Candida boidinii, could be localized within the peroxisomes. Gel-electrophoretic studies of the peroxisome fraction demonstrated that it contained only two predominant protein bands consistent with alcohol oxidase and catalase. No alcohol oxidase and catalase activity was found in mitochondria. [ABSTRACT FROM AUTHOR]

The temperature-sensitive Chinese hamster ovary cell mutant, tsH1, has been shown previously to contain a temperature-sensitive leucyl-tRNA synthetase. At the non-permissive temperature of 40°C cytosolic protein synthesis is rapidly inhibited. The protein synthesis which continues at 40°C appears to be mitochondrial, since: (a) whole-cell protein synthesis at the permissive temperature of 34°C is not inhibited by tevenel, the sulfamoyl analogue of chloramphenicol and a specific inhibitor of mitochondrial protein synthesis; however, whole-cell protein synthesis at 40°C is inhibited by tevenel. (b) Protein synthesis by isolated mitochondria from tsHl cells is not significantly inhibited at 40°C. (c) At 40°C [14C]leucine is incorporated predominantly into the mitochondrial fraction of tsH1 cells. (d) The incorporation of [`4C]leucine at 40°C into mitochondrial proteins of tsH1 cells is inhibited by tevenel but not by cycloheximide. These results suggest that the mitochondria of tsH1 cells contain a leucyl-tRNA synthetase which is different from the cytosolic enzyme. The inhibition of cytosolic, but not of mitochondrial protein synthesis in tsHl cells at 40°C allows the selective labelling of mitochondrial translation products in the absence of inhibitors. The mitochondrial translation products labelled in tsH1 cells at 40°C and at 34°C in the presence of cycloheximide have been compared by sodium dodecylsulphate - polyacrylamide gel electrophoresis. Both conditions of labelling give similar profiles. The mitochondrial translation products are resolved into two components, one with an apparent molecular weight range from 40000 to 20000 and a second with an apparent molecular weight range from 20000 to 10000. [ABSTRACT FROM AUTHOR]

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter-Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients. An isolation method was developed using Triton X-100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub-optimal Triton X-100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane. The mitochondria and peroxisomes could not be clearly separated using conventional sucrose-Ficoll density gradients. Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes ("heavy-labelling"). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact. The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen-peroxide-producing oxidases were detected. The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 µM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine. [ABSTRACT FROM AUTHOR]

The number, size, solubility in chloroform/methanol and some aspects of the formation of the components labeled by radioactive amino acids in isolated mitochondria of rat liver and Zajdela hepatoma were studied. Isolated mitochondria were labeled with radioactive amino acids under various conditions, and the distribution of radioactivity in sodium dodecylsulfate-polyacrylamide gels after electrophoresis of mitochondrial membrane fraction was analysed. 1. Isolated mitochondria of rat liver and Zajdela hepatoma incorporated radioactive amino acids almost exclusively into the membrane fraction. Electrophoretic analysis of this fraction revealed the presence of 15 distinct peaks of radioactivity with corresponding apparent molecular weights of 10 000 to 58 000. The electrophoretic mobility of the labeled components was identical and the general pattern of the radioactivity distribution in the gel for the rat liver and the tumour mitochondria was very similar. 2. Components of the membrane fraction of rat liver mitochondria labeled in vitro displayed an unequal solubility in acidic (2 mM HCl) chloroform/methanol (2/1) mixture; as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis a single labeled component with apparent molecular weight of 10 000 was soluble in neutral chloroform/methanol. 3. Inverse relation was observed between amino acid incorporation activity of isolated mitochondria and the portion of the label incorporated into the component with apparent molecular weight 10 000. The identity of this component with that soluble in neutral chloroform/methanol mixture has been indicated. 4. The rate of incorporation of [³H]leucine by isolated Zajdela hepatoma mitochondria into the components with lower (10 000-25 000) apparent molecular weights decreased with time, whereas that into components with higher (above 25 000) apparent molecular weight remained approximately constant within the time interval tested (30 min). 5. From the total radioactivity incorporated into the membrane fraction during 5-min pulse labeling of isolated Zajdela hepatoma mitochondria by [³H]leucine up to 25% was recovered in the region of the gel corresponding to a component with apparent molecular weight 10 000. After 25 min chase the radioactivity in this region decreased about 3.5 times while the specific radioactivity of the total membrane fraction did not change significantly. The pattern of radioactivity distribution observed after the pulse was preserved by chloramphenicol. 6. Unlabeled sonicated mitochondria or postribosomal supernatant from rat liver regenerating in the presence of chloramphenicol were incubated with neutral chloroform/methanol extract of in vitro with [14C]leucine labeled rat liver mitochondria. After this incubation several labeled components with apparent molecular weights above 10 000 were recovered in the electrophoreograms of the originally unlabeled fractions. The results suggest a posttranslational modification of the products of mitochondrial protein synthesis consisting either in conversion of the highly hydrophobic low molecular weight product of mitochondrial protein synthesis into components of higher molecular weight or the interaction of the former product with other proteins to increase their hydrophobicity. [ABSTRACT FROM AUTHOR]

The halothane-dependent, calcium-induced loss of respiratory control in rat liver mitochondria [1,2] is Mg2+-dependent and is accompanied by an enhanced mitochondrial swelling. It is suggested that this swelling reflects an increase in calcium activity in the matrix space, due to a decrease in binding of the accumulated cation. This change in the partition of intramitochondrial calcium is correlated with an inhibition by halothane of energy-independent, calcium-induced swelling. The enhanced swelling associated with the active accumulation of calcium in the presence of halothane does not lead to a marked increase in permeability to other ions. Nevertheless, under conditions of energised calcium uptake, and in the presence of Mg2+, a halothane-dependent, ruthenium red-insensitive efflux of calcium is observed. This is consistent with the proposed halothane- dependent increase in the matrix activity of accumulated Ca2+. It is suggested that this mechanism accounts for the previously postulated [2] futile cycle of calcium uptake and release induced by halothane in rat liver mitochondria. [ABSTRACT FROM AUTHOR]

The previously reported (Hall et al., Biochem. Soc. Trans. 1973) halothane-dependent, calciuminduced loss of respiratory control in rat liver mitochondria is relatively specific to calcium; the effect of strontium ions is much smaller, and comparable additions of potassium salts have no effect on mitochondrial respiration on succinate in the presence of halothane. The calcium-dependent loss of respiratory control can be prevented, or reversed, respectively, by the prior or subsequent addition of agents that either chelate extramitochondrial Ca2+ or inhibit calcium accumulation, or that inhibit the efflux of accumulated calcium. These results suggest that the halothane-dependent, calcium-induced loss of respiratory control is due to a cyclic flux of calcium uptake and release. [ABSTRACT FROM AUTHOR]

Yeast mitochondrial DNA labelled in vitro by incubation of isolated mitochondria with DNA precursors exhibits skewed profiles on isopycnic CsCl gradients. The skew is not due to nuclear DNA nor to single-stranded mitochondrial DNA in the product labelled in vitro. Simultaneous labelling with [³H]dTTP and [14C]dGTP in vitro indicates a gradient of base composition in the DNA labelled in vitro. Thus, selective degradation of mitochondrial DNA occurs during incubation, converting large molecules having the mean density of mitochondrial DNA into smaller molecules of higher mean density and with higher G:T ratio. Similarly skewed distributions can also be produced by incubation of mitochondrial DNA labelled in vivo with the yeast mitochondriaI fraction or with micrococcal endonuclease, an enzyme known to selectively hydrolyse (A + T)-rich regions of DNA [ABSTRACT FROM AUTHOR]

The conformation of the closed circular mitochondrial DNA in cultured human cells is changed by the addition of berenil to the culture medium in such a way that the 34-s mitochondrial DNA is converted into a 29-S DNA and finally into a 24-S DNA. The superhelix density of the covalently closed 29-S mitochondrial DNA is σ0 = -1.5 × 10-2 (turns/10 base pairs) and consequently is intermediate between the superhelix density of the 34-S DNA (σ0 = -2.8 × 10-2) and that of the closed circular 24-S DNA (σ0 ≈ 0). Removal of the drug reverses the transition, covalently closed circular 34-S DNA to covalently closed circular 29-S DNA to covalently closed circular 24-S DNA. The covalently closed circular 24-S mitochondrial DNA is not replicated. Transcription of this DNA is also inhibited as indicated by the fact that no synthesis of the mitochondrial ribosomal RNAs occurs as long as the mitochondrial DNA is in this conformation. The covalently closed circular 29-S mitochondrial DNA is replicated but not transcribed in vivo. [ABSTRACT FROM AUTHOR]

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP → ATP + creatine) equal to 3.5 µmol × min-1 × mg-1 is essentially higher than maximal rate of the forward reaction (0.8 µmol × min-1 × mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in a creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reaction mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be explained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP and ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase. [ABSTRACT FROM AUTHOR]

Six types of nuclease activities were found to be concentrated in the large granule fraction isolated from rat liver homogenates by differential centrifugation. Analysis by density equilibration shows that three nucleases are associated with mitochondria: an alkaline ribonuclease (pH optimum 8.8), an alkaline deoxyribonuclease (pH optimum 7.6) and an enzyme acting on polyriboadenylate (pH optimum 7.5). When the outer mitochondrial membrane is ruptured in hypotonic medium, the three mitochondrial nucleases are partially solubilized. Solubilization is however obtained by addition of KCl to the suspension medium. It is concluded that mitochondrial nucleases are localized in the intermembrane space but that an adsorption to the outer face of the inner mitochondrial membrane occurs in sucrose 0.25 M. The mitochondrial localization of alkaline ribonuclease, alkaline deoxyribonuclease and polyadenylate accounts for at least 80% of the activity of liver homogenate; nevertheless, an excess of these enzymes is present in the microsomal fraction. Although no definitive conclusion can be reached for the significance of this observation, it is shown by density equilibrium analysis that these nucleases are not associated either with ribosomes or with the membranes which are the major component of the microsomal fraction. [ABSTRACT FROM AUTHOR]

The initial rates of the exchange external oxoglutarate/internal malate through the inner membrane of rat-heart mitochondria, for various concentrations of the two substrates, have been reinvestigated for an extended range of concentrations of the external oxoglutarate. This has been made possible by use of the inhibitor-stop technique that allows 100 times smaller incubation times than the centrifugation-stop technique used previously. Under the experimental conditions the uptake of the external-labelled oxoglutarate into the mitochondrial-matrix space is mediated by the oxoglutarate translocator performing a ono-to-one exchange of the anions oxoglutarate (external) and malate (internal). Two intermediary-plateau regions are observed in the kinetic saturation curve of the translocator by the external oxoglutarate, revealing a complex rate equation which is found to be the product of two one-substrate functions. Analysing these features it is shown that the model, proposed earlier, of a ‘double carrier’ as catalyst in a rapid-equilibrium random bi-bi mechanism, is still applicable but that several external binding sites have to be considered. As already noticed the external and the internal substrates bind to their respective sites independently of each other. Furthermore, some additional requirements imposed by the observed kinetics suggest that the exchange reaction is performed by only one translocator species made of identical interacting subunits. The anion exchange is tentatively viewed as a rotation of a subunit around an axis situated in the plane of the membrane after two independent local configuration changes induced by the binding of the two substrates on this subunit. [ABSTRACT FROM AUTHOR]

This report describes a kinetic analysis of energy-linked Ca2+ transport in rat liver mitochondria, in which a ruthenium red/EGTA [ethanedioxy-bis(ethylamine)-tetraacetic acid] quenching technique has been used to measure rates of45Ca2+ transport. Accurately known concentrations of free 54Ca2+ were generated with Ca2+/nitrilotriacetic acids buffers for the determination of substrate/velocity relationships. The results show that the initial velocity of transport is a sigmoidal function of Ca2+ concentration (Hill coefficient = 1.7), the Km being 4 μM Ca2+ at 0 °C and pH 7.4. These values for the Hill coefficient and the Km remain constant in the presence of up to 2 mM phosphate, but with 10 mM acetate both parameters are increased slightly. Both permeant acids increase the maximum velocity to an extent dependent on their concentration. The Ca2+-binding site(s) of the carrier contains a group ionizing at pH approximately 7.5 at 0 °C, which is functional in the dissociated state. The stimulatory effect of permeant acids is ascribed to their facilitating the release of Ca2+ from the carrier to the internal phase, an interpretation which is strengthened by the lack of effect of the permeant anion SCN¯ on Ca2+ transport. Studies on the time-course of Ca2+ uptake and of EGTA-induced Ca2+ efflux from pre-loaded mitochondria demonstrate the reversibility of the carrier in respiring mitochondria and the extent to which this property is influenced by permeant acids. These data are accommodated in a carrier mechanism based on electrophoretic transport of Ca2+ bound to pairs of interacting acidic sites. [ABSTRACT FROM AUTHOR]

In a model system consisting of highly coupled rat liver mitochondria respiring in the presence of substrate, pyruvate kinase, phosphoenolpyruvate, ATP, hexokinase and glucose, the increase in the mitochondrial concentration results in a progressive decrease in the activity of pyruvate kinase. These results are in accord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with mitochondrial oxidative phosphorylation for the available ADP. The addition of adequate amounts ofthe amino acids, cysteine, alanine and phenylalanine, known as inhibitors of pyruvate kinase, to living Ehrlich ascites tumor cell suspensions results in a stimulation ofthe respiratory rate and in a decrease of the glycolytic rate ofthe cells. Concomitant with these changes, there is an accumulation of intracellular phosphoenolpyruvate and ADP, and a decrease in pyruvate and ATP. These results provide additional evidence for paying attention to pyruvate kinase as another key enzyme whose properties and activities may be major determinants for the control of glycolysis and the Crabtree and Pasteur effects of tumor cells. [ABSTRACT FROM AUTHOR]

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of ‘sonic’ submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 μM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 μM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malate and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site 1 phosphorylation to octylguanidine. [ABSTRACT FROM AUTHOR]

Mitochondrial proteins soluble in neutral chloroform-methanol (2:1) were separated from lipids by ether precipitation and resolved by Sephadex G-200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak II). Residual phospholipids are found only in peak II. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid. Cycloheximide stimulates almost two-fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak II proteins by 95 %. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70 % and 95 % respectively, but do not affect labelling of peak II proteins. At least 30 % of the translation products of mitochondrial DNA in vitro behave like peak I proteins: they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation. The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak II are encoded by nuclear genes and synthesized on cytoplasmic ribosomes. Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat-stable aggregates in the presence of dodecylsulfate. [ABSTRACT FROM AUTHOR]

1.12-(9-Anhroyl)stearic acid can be incorporated into mitochondrial membranes. 2. The fluorescence properties of the membrane-bound probe are different from those of the free molecule. 3. The intensity of emission and fluorescence life-time of the probe is enhanced when, in the presence of substrate, the electron-transport chain is reduced. 4. This change in intensity has been demonstrated to be a result of collisional quenching by oxidised ubiquinone in the oxidised membrane but not when the respiratory chain is in the reduced state. 5. In pulsing anaerobic mitochondria with oxygen the rate of the fluorescence change is found to be slower than the rate ubiquinone oxidation, suggesting that the probe detects a structural transition in the mitochondrial inner membrane. 6. This transition results in a constant on ubiquinone motion in the reduced system. Model experiments, using lipid dispersions, have been carried out to test some of the interpretations. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA from Euglena gracilis has been investigated in its chemical and physical properties. Its G + C content is equal to 25 %; its buoyant density in a CsCl density gradient (1.690 g/ cm³) is higher, by 5 mg/cm³, than expected for a bacterial DNA having the same base composition. The buoyant densities of denatured and renatured DNA are higher than that of native DNA by 10-12 mg/cm³ and 6 mg/cm³, respectively. The melting temperature, Tm, is 77 °C in standard saline citrate; the first derivative of the melting curve shows a striking multimodality. Degradation of the DNA by micrococcal nuclease indicates that about 40% of the DNA is formed by stretches lower than 10% in G + C. In all its properties the mitochondrial DNA from Euglena gracilis is strikingly similar to that of Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

When mitochondrial ATPase, which has been modified on a single tyrosine residue by 4-chloro-7-nitrobenzofurazan, is incubated at pH 9.0, the 7-nitrobenzofurazan group undergoes an intra-molecular transfer to a nitrogen residue. The rate of this transfer is sensitive to the binding of adenine nucleotides to the enzyme. The resulting N-nitrobenzofurazan ATPase has little or no activity. The fluorescence of the N-nitrobenzofurazan group in the modified ATPase is quenched on binding of ADP. Electrophoresis of the modified enzyme in sodium dodecyl sulphate on a 10% polyacrylamide gel shows that the fluorescence of the N-nitrobenzofurazan chromophore is exclusively in the/3 subunit. The rate of transfer of the nitrobenzofurazan group from tyrosyl oxygen to nitrogen on the enzyme is compared with the rate of transfer between model compounds. The interaction of the N-nitrobenzofurazan ATPase with aurovertin is reported. [ABSTRACT FROM AUTHOR]

Soluble mitochondrial ATPase (F1) isolated from Neurospora crassa is resolved by dodecyl-sulfate-gel electrophoresis into five polypeptide bands with apparent molecular weights of 59000, 55 000, 36 000, 15 000 and 12 000. At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X-100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F1 ATPase. Two of the associated polypeptides with apparent molecular weights of 19000 and 11000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leucine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial (cycloheximide) protein synthesis. The appearance of mitochondrial translation products in the immunoprecipitated ATPase complex is inhibited by cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the ATPase complex. [ABSTRACT FROM AUTHOR]

Pulse-labeled replicating mitochondrial DNA molecules of three different cell lines, mouse NCTC 929 (ATCC; CCL 1), monkey Vero (ATCC;CCL 81) and human WISH (ATCC; CCL 25) were classified according to their sedimentation velocity in neutral sucrose gradients. Replicating molecules were isolated and the newly synthesized strands were dissociated from the parental DNA strands. This procedure yielded parental closed circular duplex DNA with a higher sedimentation velocity in neutral sucrose gradients and higher absolute values of superhelix density in comparison with values for mature unreplicating supercoiled mitochondrial DNA. Replicating molecules with these properties, i.e. higher absolute superhelix densities of the parental strands, comprise 2-4% of the total mass of the mitochondrial DNA in all three cell lines. In addition, mouse cells contain a large proportion of D-loop mitochondrial DNA molecules (28 S). Upon dissociation of the 8-S single-stranded initiation sequence from the parental stroids, these molecules yielded parental supercoiled duplex DNA with a superhelix density similar to that of mature supercoiled mitochondrial DNA. D-loop mitochondrial DNA represents about 30% of the total mass of the mitochondrial DNA in mouse cells. Therefore, two populations of replicating mitochondrial DNA can be distinguished in mouse cells. One of these accumulates as D-loop mitochondrial DNA with constant topological winding, while the other replicates in a manner similar to that of Vero and WISH cells under progressively decreasing topological winding of the parental strands. [ABSTRACT FROM AUTHOR]

Closed-circular DNA has been isolated from mitochondrial-enriched fractions from four 'petitenegative' yeasts. Electron microscope analysis has shown in each case the presence of a large discrete size Class of circular DNA greater than 6 μm in length and smaller heterodisperse circular DNA less than 6 μm. Length and molecular weight measurements of the large circular DNA are: Candida parapsilosis, 11.14 ± 0.45 μm and 23.1 x 106; Hansenula wingei, 8.22 ± 0.43 μm and 17.3x 106; Kluyveromyces lactis, 11.44 ± 0.20 μm and 24.0 x 106 and Schizosaccharornyces pombe, 6.04 ± 0. 16 μm and 12.5 x 106. These circular DNAs are thought to be mitochondrial DNA on the basis of their buoyant densities and their enrichment with mitochondria. The finding of a gross size difference between mitochondrial DNA from petite-negative yeasts and Saccharomyces cerevisiae focuses attention on the suggestion that certain unique properties of the latter organism's mitochondrial DNA may be of relevance to the mechanism of the petite mutation in yeast. [ABSTRACT FROM AUTHOR]

Isolated rat liver mitochondria incorporate [14C]nicotinamide into NAD. Nicotinic acid and quinolinic acid are not utilized. The incorporation of nicotinamide into NAD is stimulated by ATP. After incubation of the mitochondria with [14C]nicotinamide, labeled NMN can be isolated. No labeling of nicotinate mononucleotide or nicotinate adenine dinucleotide could be observed. Azaserine has no effect on the incorporation of nicotinamide into NAD. Labeled NAD is also obtained after addition of [14C]ATP to the mitochondria. The incorporation of radioactivity from extramitochondrial [14C]ATP into NAD is inhibited by carboxyatractyloside. On the basis of these results, it is concluded that the mitochondria incorporate nicotinamide by an intramitochondrial biosynthetic pathway via NMN. [ABSTRACT FROM AUTHOR]

35S-labelled atractylate and carboxy-atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP. ATP carrier in beef heart mitochondria. The following results are obtained. 1. Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier. 2. The maximum number of binding sites of both atractylates is about 1.6 μmol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd = 5 · 10–8 M and for carboxy-atractylate Kd = 10–8 M. The mass action plots of the concentration dependence for the binding are nonlinear-convex in particular with carboxy-atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding. 3. The binding of atractylate and carboxy-atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion. 4. The atractylates remove ADP in a nearly 1:1 stoichiometry from untreated mitochondria. In aged and Pi-treated membranes the ratio ΔADP/Δatractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations then that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP. 5. Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding, The removal of atractylate depends on the synergistic effect of bongkrekate with ADP. 6. The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside. [ABSTRACT FROM AUTHOR]

35S-labelled atractylate and carboxy-atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP. ATP carrier in beef heart mitochondria. The following results are obtained. 1. Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier. 2. The maximum number of binding sites of both atractylates is about 1.6 μmol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd = 5 · 10–8 M and for carboxy-atractylate Kd = 10–8 M. The mass action plots of the concentration dependence for the binding are nonlinear-convex in particular with carboxy-atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding. 3. The binding of atractylate and carboxy-atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion. 4. The atractylates remove ADP in a nearly 1:1 stoichiometry from untreated mitochondria. In aged and Pi-treated membranes the ratio ΔADP/Δatractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations then that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP. 5. Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding, The removal of atractylate depends on the synergistic effect of bongkrekate with ADP. 6. The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside. [ABSTRACT FROM AUTHOR]

The macrolide-type antibiotic chlorothricin was found to inhibit both the mitochondrial and the cytoplasmic form of pig heart malate dehydrogenase, Steady-state kinetic measurements revealed that in the direction of oxalacetate reduction chlorothricin is competitive with respect to NADH and non-competitive with respect to oxalacetate. Both the variation of initial velocity with NADH concentration in the presence of antibiotic, and, at several fixed levels of NADH, the variation of initial velocity with chlorothricin concentration deviates from the classical Michaelis­Menten relationship for the two isoenzymes. Since, despite the very similar kinetic behaviour of the mitochondrial and cytoplasmic species of malate dehydrogenase, the concentration of chlorothricin required for half-maximal inhibition of the two enzymes differs by more than a factor of 10 (the mitochondrial isoenzyme being more susceptible to inhibition), it is concluded that the NADH binding sites of the mitochondrial and cytoplasmic form of malate dehydrogenase from pig heart are different. [ABSTRACT FROM AUTHOR]

1. In order to demonstrate that triiodothyronine affects mitochondrial RNA synthesis by acting on the enzyme component of the DNA · RNA polymerase complex, mdtochondrial RNA polymerase from thyroidectomized and hormone-treated rats was purified up to a stage in which activity was dependent on the addition of exogenous template. In these conditions and using different DNAs as templates, the enzyme from hormone-treated animals displayed an activity about double that of the activity of thyroidectomized animals. 2. Measurements of stability of mitochondrial RNA synthesized in vitro suggest, however, that the hormone can act also at the template level in mitochondrial transcription: the RNA population synthesized in vitro from hormone-treated rats is indeed much more enriched in unstable, probably messenger, RNA species. 3. The turnover of mitochondrial messenger RNA is higher after hormone treatment. 4. Adenosine cyclic 3′: 5′-monophosphate (cAMP) and its dibutyryl derivative added in vitro to mitochondria from thyroidectomized animals do not affect the incorporation of labeled precursor into mitochondrial RNA, suggesting that the level of the cyclic nucleotide in mitochondria is probably not involved in the hormone action. 5. It is concluded from these and previous studies that the thyroid hormone affects more than one parameter in the mitochondrial transcription process. The interrelationship between these events at molecular level remains, however, to be clarified. [ABSTRACT FROM AUTHOR]

Venturicidin is a specific inhibitor of aerobic growth of yeast and has no effect on fermentative growth, a result which is consistent with its known mode of action on mitochondrial oxidative phosphorylation. Venturicidin-resistant mutants of Saccharomyces cerevisiae have been isolated and form two general classes: class 1, nuclear mutants which are resistant to a variety of mitochondrial inhibitors and uncouplers, and class 2, mitochondrial mutants of phenotype VENR OLYR and VENR TETR in vivo. VENR OLYR mutants show a high degree of resistance to ventuficidin and oligomycin at the whole cell and mitochondrial ATPase level but, in contrast, no resistance at the mitochondrial level is observed with VENR TETR mutants. Venturicidin resistance/sensitivity can be correlated with two binding sites on mitochondrial ATPase, one of which is common to the oligomycin binding site and the other is common to the triethyl tin binding site. Biochemical genetic studies indicate that two mitochondrial genes specify venturicidin resistance/sensitivity and that the mitochondrial gene products are components of the mitochondrial ATPase complex. [ABSTRACT FROM AUTHOR]

Mutants of Saccharomyces cerevisiae resistant to triethyl tin sulphate have been isolated and are cross-resistant to other trialkyl tin salts. Triethyl-tin-resistant mutants fall into two general phenotypic classes: class I and class 2. Class 1 mutants are cross-resistant to a variety of inhibitors and uncoupling agents which affect mitochondrial membranes (oligomycin, ossamycin, valinomycin, antimycin, erythromycin, chloramphenicol, ‘1799’, tetrachlorotrifluoromethyl benzimidazole, carbonylcyanide-m-chlorophenylhydrazone and cycloheximide). Class 2 mutants are specifically resistant to triethyl tin and the uncoupling agent ‘1799’ [bis-(hexafluoroacetonyl)-acetone]. Triethyl tin at neutral pH values is a specific inhibitor of mitochondrial energy conservation reactions and prevents growth on oxidisable substrates such as glycerol and ethanol. Triethyl-tin-resistant mutants grow normally on glucose and ethanol in the presence of triethyl tin (10 μM). Biochemical studies indicate that the mutation involves a modification of the triethyl tin binding site on the mitochondrial inner membrane, probably the ATP-synthetase complex. Triethyl tin resistance/sensitivity in yeast is determined by cytoplasmic (mitochondrial) and nuclear genes. The mutants fall into a nuclear and a cytoplasmic (mitochondrial) class corresponding to the phenotypic cross-resistance classes 1 and 2. In the cytoplasmic mutants the triethyl tin resistance segregates mitotically and the resistance determinant is deleted by the action of ethidium bromide during petite induction. Recombination studies indicate that the triethyl tin mutations are not allelic with the other mitochondrial mutations at the loci RI, RIII and OLI. This indicates that the binding or inhibitory sites of oligomycin and triethyl tin are not identical and that the triethyl tin binding site is located on a different mitochondrial gene product to those which are involved in oligomycin binding. Interaction and cooperative effects between different binding sites on the mitochondrial inner membrane have been demonstrated in studies of the effect of the insertion of the TETR phenotype into mitochondrial oligomycin-resistant mutants and provide an experimental basis for complementation studies at the ATP-synthetase level. [ABSTRACT FROM AUTHOR]