DIRECT COUNTING AND SIZING OF MITOCHONDRIA IN SOLUTION

Resistive particle counting has been developed for the accurate sizing and counting of mitochondria in solution. The normal detection limit with a 30 µ aperture is 0.48 µ diameter, or 0.056 µ³ particle volume The mean volume of rat liver mitochondria was 0.42 µ³ or 0.93 µ in diameter. The average value for numbers of particles per milligram of mitochondrial protein was 4.3 x 10³, and per gram of rat liver was about 11 x 10¹⁰. These values compare satisfactorily with those derived by light microscopy and electron microscopy. The mean volume for mitochondria from rat heart was 0 60 µ³ and from rat kidney cortex, 0.23 µ³. These values agree within 15% of those determined by electron microscopy of whole tissue. Mitochondrial fragility and contaminating subcellular organelles were shown to have little influence on the experimentally determined size distributions The technique may be applied to rapid swelling studies, as well as to estimations of the number and size of mitochondria from animals under different conditions such as liver regeneration and hormonal, pathological, or drug-induced states Mitochondrial DNA, RNA, cytochrome c-oxidase, cytochrome (a ÷ a₃), and iron were nearly constant per particle over large differences in particle size. Such data may be particularly valuable for biogenesis studies and support the hypothesis that the net amount per particle of certain mitochondrial constituents remains constant during mitochondrial growth and enlargement     

SUBCELLULAR MORPHOMETRIC AND BIOCHEMICAL ANALYSES OF DEVELOPING RAT HEPATOCYTES

Livers of rats between the 16th gestational and 100th postnatal day of age were subjected to quantitative biochemical and electron microscope, morphometric analyses. The amount of total mitochondrial protein per gram of liver remained at 34% of the adult level throughout the last 4 days of gestation but this was the period of rapid rise in the levels of cytochrome c oxidase, aspartate aminotransferase, and glutamate dehydrogenase in mitochondria; the nuclear fraction also acquired some glutamate dehydrogenase but lost most of it during postnatal development. During early postnatal life the amount of mitochondrial protein rose in parallel with the levels of cytochrome c oxidase and glutamate dehydrogenase but the upsurges of glutaminase and, later, of ornithine aminotransferase were accompanied by relatively little change in total mitochondrial protein. The surface area of rough endoplasmic reticulum per unit volume of hepatocyte cytoplasm (S_v^RER) did not change significantly throughout the period of development studied. From the 16th day of gestation to term the surface area of smooth ER (S_v^SER), the volume occupied by mitochondria (V_v^MT) and their number (N_v^MT) remained at 30, 66, and 45% of their adult values, respectively. V_v^MT and N_v^MT attained their maximal levels by the 2nd postnatal day and S_v^SER between days 2 and 12. Mitochondria of adult liver are thus smaller and contain more protein per unit volume than do those of fetal liver. After the 12th postnatal day, hepatocytes treble their size; they acquire more cytoplasm with additional enzymes but without further change in organelle concentration. The data reveal several distinct phases in the differentiation of hepatocytes. Each phase can be characterized by the extent to which the quantity and composition of various subcellular compartments evolve.     

Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1. Rats were injected with [U-¹⁴C]glucose and the content of ¹⁴C in proteins and lipids of the cerebral P₁ (`nuclear'), P<sub>2</sub> (`mitochondrial'), P₃ (`microsomal') and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P<sub>2</sub>) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the <sup>14</sup>C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the <sup>14</sup>C assimilated by brain proteins was found in the P<sub>2</sub> (`mitochondrial') fraction. About 68–70% of the ¹⁴C assimilated by brain lipids was also recovered from the lipids of the P₂ fraction. 4. Between 22 and 93hr. after injection of [U-¹⁴C]glucose both the amount of ¹⁴C in the protein of the P₂ (`mitochondrial') fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P₁, P₂ and P₃) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P₂) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U-¹⁴C]glucose. The amount of ¹⁴C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of ¹⁴C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U-¹⁴C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.     

Purification of B. megatherium Phage G and Evidence for a Muralytic Enzyme as an Integral Part of the Phage

The Purification Of B. megatherium G phage is described and it is shown that DEAE cellulose chromatography combined with conventional methods gave a phage preparation which was at least 95 per cent pure, and contained 2.16 µg nitrogen/10¹¹ infective particles. The phage particle weight in molecular weight units was 91 x 10⁶. The small amount of contaminating material appeared to represent phage "ghosts." An essentially 1:1 ratio of particles to infective units was found when data from electron microscopic counts or data from chemical analysis were related to phage infectivity. Comparison, by several methods, of the G phage and coliphage T2 shows that T2 is 2.6 times larger than G phage. The specific activity of the muralytic component obtained by disintegration of phage preparations with urea was unchanged by the purification indicating that the phage-"bound" muralytic activity is an integral part of the phage structure.     

Effects of neuroleptics on 3H-haloperidol and 3H-cis(Z)-flupenthixol binding and on adenylate cyclase activity in vitro.

The subcellular localization of dopamine-sensitive adenylate cyclase was studied in rat brain striatum and compared to the distribution of dopamine binding sites. The highest specific activity of adenylate cyclase activities sensitive to dopamine was associated almost exclusively with synaptic membranes (mithchondrial fraction; P₂). Using [³H] haloperidol and [³H] apomorphine as markers for the dopamine receptor, specific binding was observed in both the mitochondrial (P₂) and microsomal (P₃) fractions. Data for the mitochondrial fraction revealed a heterogeneity of binding sites. Two saturable sites for [³H] haloperidol were observed with K_d values of 2.5nM and 12.5nM respectively. Overall, the localization of multiple binding sites in the crude synaptosomal fraction correlates well with the localization of dopamine-sensitive adenylate cyclase in this fraction.     

DNA from plant mitochondria

DNA was isolated from a mitochondrial fraction of each of the following plant materials: Mung bean (Phaseolus aureus) etiolated hypocotyl; turnip (Brassica rapa) root; sweet potato (Ipomoea batatas) root; and onion (Allium cepa) bulb. It was found that all of these mitochondrial fractions contained DNA, the densities of which were identical (ρ=1.706 g·cm⁻³). An additional DNA (ρ=1.695) band found in the mitochondrial fraction of Brassica rapa, was identical to DNA separately isolated from the chloroplast-rich fraction. The origin of the second DNA from Allium mitochondrial fraction was not identified.

Contrary to the identity of the mitochondrial DNA, DNA from nuclear fractions differed not only with each other but from the corresponding mitochondrial DNA.

DNA from Phaseolus and Brassica mitochondria showed the hyperchromicity characteristic of double stranded, native DNA upon heating; Tm's in 0.0195 Na⁺ were the same; 72.0°. The amount of DNA within the mitochondrion of Phaseolus was estimated to be 5.0 × 10⁻¹⁰ μg; this estimate was made by isolating the mitochondrial DNA concomitantly with the known amount of added ¹⁵N²H B. subtilis DNA (ρ=1.740). Approximately the same amount of DNA was present in the mitochondrion of Brassica or Ipomoea.

     

Variation in protein lateral diffusion coefficients is related to variation in protein concentration found in mitochondrial inner membranes

The electrophoretic freeze-fracture electron microscopy method (Sowers, A.E. and Hackenbrock, C.R. (1984) Proc. Natl. Acad. Sci. USA 78, 6246–6250) for measuring the lateral diffusion coefficient of integral proteins was applied to a large population of spherical-shaped mitochondrial inner membranes. Membrane integral protein concentration was estimated by determining the intramembrane particle concentration. Analysis of the data reveals that: (a) the radii of the spherical inner membranes in the selected population ranged from 0.22 to 1.2 μm, (b) the intramembrane particle concentrations ranged from 2300 to 6400 per μm², and (c) the calculated lateral diffusion coefficients of the intramembrane particles ranged from 1.3·10⁻¹⁰ to 3.35·10⁻⁹ cm²/s. The data clearly show a naturally occurring large range in protein concentration in the mitochondrial inner membrane and an inverse correlation of lateral diffusion coefficient with the membrane protein concentration. This study is the first to show that the lateral diffusion coefficient of integral proteins in a native membrane varies as the membrane protein concentration.     

Highly enriched,minimally disrupted plasma membrane vesicles from aortic myocytes grown in primary culture

A plasma membrane-enriched fraction (fraction 1B) has been obtained from rat aortic myocytes grown in primary culture. Plasma membrane markers, 5′-nucleotidase and ouabain-sensitive (Na⁺ + K⁺)-ATPase, are enriched 4.1- and 8.7-fold, respectively, in this fraction. Although endoplasmic reticulum marker NADPH-cytochrome c reductase is the most enriched in mitochondrial and heavy sucrose density gradient fractions, substantial enrichment of this marker is also observed in membrane fraction 1. This membrane preparation therefore contains a certain quantity of endoplasmic reticulum. Cytochrome c oxidase is de-enriched by a factor of 0.04 in fraction 1, indicating that it is essentially clear of mitochondrial contamination. Homogenization of aortic media-intima layers using a whole-tissue technique induces greater disruption of mitochondria and subsequent contamination of membrane fractions than does the procedure for cell disruption. Analysis of electrophoretic gels, vesicle density distribution and electron micrographs of enriched membrane fractions provide evidence that plasma membrane enriched from cultured myocytes is less traumatized than comparable fractions obtained from intact tissue. The potential value of such a highly enriched, minimally disrupted plasma membrane preparation is discussed.     

Adenosine triphosphatases of cestodes Bothriocephalus scorpii

Activities and properties of adenosine triphosphatases (ATPases) were studied in mitochondrial and microsomal fractions of cestodes Bothriocephalus scorpii parasitizing in pyloric appendages of the Brandt’s bullhead Myoxocephalus brandti. The highest activity was revealed in the mitochondrial fraction. The mitochondrial and microsomal fractions of B. scorpii had the ATPase activity dependent on the presence of cations Mg²⁺, Mn²⁺, and Ca²⁺. Effects of ions and inhibitors on the B. scorpii ATPase activity with various cations were. Both subcellular fractions were able to hydrolyze, apart from ATP, also GTP, CTP, and UTP.     

Subcellular Localization of Angiotensin I Converting Enzyme in Rabbit Lung

THE rapidity with which angiotensin I is converted to angiotensin II^1–5 suggests that the subcellular localization of the converting enzyme is important. Bakhle⁶ and Cushman and Cheung⁷ have demonstrated the particulate nature of this enzyme. Subcellular distribution studies and marker enzyme analysis indicate that converting enzyme activity in rabbit lung is most concentrated in the pellet sedimenting at between 1,000 and 25,000g (P₂), subsequently characterized as the light and heavy mitochondrial fraction. To identify this fraction more fully, we have resuspended P₂ and centrifuged it through a discontinuous density gradient, a procedure which separates converting enzyme activity from the mitochondria. Marker enzyme analysis and electron microscopy suggest plasma membrane as the major constituent of that fraction displaying highest specific activity of converting enzyme.     

Origin of acetate in spinach leaf cell

Mitochondria were isolated from spinach (Spinacia oleracea L.) leaves using a Percoll gradient step. The high purity of the organelle fraction is demonstrated by electron microscopy and biochemical parameters. In the matrix space of these mitochondria, a short-chain acyl-coenzyme A hydrolase is present that converts acetyl-coenzyme A to acetate and coenzyme A with reasonable rates (K_m, 150 micromolar; V_max, 140 nanomoles acetate formed milligram¹ protein hour⁻¹). The enzyme is product inhibited by coenzyme A-sulfhydryl, other thiols are ineffective; however, the disulfides 5,5′-dithio-bis-(2-nitrobenzoate) and cystamine stimulate the hydrolysis. The possible role of this mitochondrial enzyme as a means of generating free acetate from pyruvate via acetyl-coenzyme A in the mitochondria of mature spinach leaves is discussed. It is suggested that free acetate moves rapidly from the mitochondrion to the chloroplast where acetyl-coenzyme A synthetase, solely localized in this organelle, converts the metabolically inert free acetate to the highly active acetyl-coenzyme A.     

A Plastidial Localization and Origin of l-Glutamate Dehydrogenase in a Soybean Cell Culture

The subcellular distribution of l-glutamate dehydrogenase (GDH, EC 1.4.1.3.) was studied in SB3 soybean (Glycine max) cells using subcellular fractionation techniques. Compounds that inhibit protein synthesis either on 80s or 70s ribosomes were also used to give a preliminary idea of which subcellular fraction is involved in GDH synthesis. It was found that whereas cycloheximide and puromycin considerably reduced the total amount of protein synthesized by the cells, they did not appear to inhibit the synthesis of GDH. In the presence of chloramphenicol, both GDH activity and protein level in the cells were considerably reduced, suggesting that this enzyme was synthesized in organelles and not in the cytosol. Streptomycin, which inhibits plastid protein synthesis, also inhibited synthesis of GDH, indicating that a fraction of GDH activity was plastidial in origin. This is supported by the data on subcellular distribution of the enzyme, which showed that a major fraction of GDH is found in the plastidial fraction, although some activity is found associated with the mitochondrial fraction also. Since a major fraction of GDH activity was found in the plastidial fraction, we studied protein synthesis using isolated plastids and ³⁵S-methionine. Using antibodies raised against purified GDH, we identified a ³⁵S-labeled 41-kilodalton polypeptide synthesized by plastids as GDH.     

CYTOPLASMIC DNA SYNTHESIS IN AMOEBA PROTEUS : III. Further Studies on the Nature of the DNA-Containing Elements

The application of electron microscope autoradiography to Amoeba proteus cells labeled with tritiated thymidine has permitted the identification of morphologically distinct particles in the cytoplasm as the sites of incorporated DNA precursor. The particles correspond to those previously described from light microscope studies, with respect to both H³Tdr incorporation and distribution in centrifugally stratified amoebae. Ingested bacteria differ from the particles, in morphology as well as in the absence of associated label. Attempts to introduce a normal particle labeling pattern by incubating amoebae with labeled sediment derived from used amoeba medium failed. The resultant conclusion, that the particles are maintained in the amoeba by self-duplication, is supported by the presence of particles in configurations suggestive of division.     

Effect of Particle Shape on Dry Particle Inhalation: Study of Flowability, Aerosolization, and Deposition Properties

The shape effects of dry particles on flowability, aerosolization, and deposition properties in inhalation drug delivery are studied. The properties are compared with similar size range particles of different shapes such as sphere, needle, cube, plate, and pollen. Flowability of the particles is characterized by Carr’s compressibility index and angle of slide (θ) method. The aerosolization and deposition properties of the particles are studied in vitro using an eight-stage Anderson cascade impactor with a Rotahaler®. Pollen-shaped particles are found to exhibit better flowability, higher emitted dose, and higher fine particle fraction than particles of other shapes in similar size range. They showed minimum θ of 35° and maximum emitted dose of 87% and fine particle fraction of 16%. The use of pollen-shaped particles can be a potential improvement in dry particle inhalation.     

AMPK-dependent and -independent coordination of mitochondrial function and muscle fiber type by FNIP1

Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1^Tg) mice. Fnip1^Tg mice were crossed with Fnip1-knockout (Fnip1^KO) mice to generate Fnip1^TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness.     

On the presence of a nicotinamide nucleotide transhydrogenase in mitochondria from potato tuber

Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD⁺ or 3-acetylpyridine-NAD⁺ by NADPH, which showed a maximum of about 50 to 150 nanomoles/minute·milligram protein at pH 5 to 6. The K_m values for 3-acetylpyridine-NAD⁺ and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total trans-hydrogenase activity.     

NUCLEAR GENE DOSAGE EFFECTS ON MITOCHONDRIAL MASS AND DNA

In order to assess the effect of nuclear gene dosage on the regulation of mitochondria we have studied serial sections of a set of isogenic haploid and diploid cells of Saccharomyces cerevisiae, growing exponentially in the absence of catabolite repression, and determined the amount of mitochondrial DNA per cell. Mitochondria accounted for 14% of the cytoplasmic and 12% of the total cellular volume in all cells examined regardless of their ploidy or their apparent stage in the cell cycle. The mean number of mitochondria per cell was 22 in the diploid and 10 in the haploids. The volume distribution appeared unimodal and identical in haploids and diploids. The mitochondrial DNA accounted for 12.6 ± 1.2% and 13.5 ± 1.3% of the total cellular DNA in the diploid and haploid populations, respectively. These values correspond to 3.6 x 10^-15 g, 2.2 x 10⁹ daltons, or 44 genomes (50 x 10⁶ daltons each) per haploid and twice that per diploid cell. On this basis, the average mitochondrion in these cells contains four mitochondrial genomes in both the haploid and the diploid.     

POLARIZATION AND ELECTRON MICROSCOPE STUDY OF FROG NERVE AXOPLASM

1. The submicroscopic organization of nerve axons from R. pipiens and R. catesbiana has been studied by means of polarizing and electron microscopes. 2. In measurements on a series of 85 fresh myelinated axons from which the sheaths had been removed average values were obtained for the total birefringence, +2.5 x 10^–4, the form birefringence, +1.4 x 10^–4, and the refractive index of the oriented component, 1.523. The average partial volume occupied by axially oriented filaments was computed to be 0.69 per cent. 3. Electron micrographs of fixed myelinated axons demonstrate an average of 93 axially oriented neuroprotofibrils per square micron of cross-section. The neuroprotofibrils are approximately 90 A in diameter, of indefinite length, and occupy a computed partial volume of 0.59 per cent. 4. Mitochondria, neuroprotofibrils, endoplasmic reticulum, and dense particles are seen in electron micrographs of both myelinated and unmyelinated nerve axons. 5. It is concluded that the neuroprotofibrils are present in the living nerve, that they play an important but undetermined role in nerve function, and that these structures are not an artifact of osmium tetroxide fixation.     

Isolation and Characterization of an Escherichia coli Bacteriophage Requiring Cell Wall Galactose

A new coliphage, designated U3, has been selected for the ability to discriminate the presence of galactose in the cell wall of Escherichia coli. U3 attacks E. coli K-12 cells that are able to incorporate galactose into their cell walls, but mutants blocked in the synthesis of uridine diphosphogalactose, the precursor of cell wall galactose, are completely resistant to the phage. U3 is a small, tail-less, approximately spherical phage resembling X174 in its physical properties. Its diameter by electron microscopy is 21 to 22 nm, and its particle weight is approximately 4 × 10⁶ daltons. Like X174, U3 appears to have a single-stranded deoxyribonucleic acid genome and has at least four cistrons.     

Exclusion of Protein from High Polymer Media: I. Derivation of Probability Distribution for the Number of Fiber Centers Within Any Sphere of Radius r

Ogston's (1958) fiber model based on Poisson's distribution function gives the average number of fibers making contact and no contact inside a sphere of radius r. The probability of penetration of spherical particles within a fibrous network was derived from the moment generating function [Formula: see text] A is the number of particles that intrude into a sphere of radious r. α(μ) is the probability that a particle, whose center is μ units away from the origin, intrudes into a sphere of radius r. A has a Poisson distribution with a mean value E(A) = 4πνα(μ)μ² dμ. The theoretical derivation of the distribution function of A gives Ogston's fiber model.