Microincineration, electron microscopy, and electron diffraction of calcium phosphate-loaded mitochondria.

Isolated rat liver mitochondria were incubated in vitro under conditions supporting the massive accumulation of calcium and phosphate. Samples were embedded, thin sectioned, and examined in the electron microscope. The intramitochondrial distribution of insoluble or structure-bound mineral substances was studied by electron microscopy coupled with recently developed techniques of high resolution microincineration. As shown previously, the ion-loaded mitochondria acquire large, internal granules which have inherent electron opacity indicative of high mineral content. Study of ash patterns in preselected areas of sections directly confirmed the high mineral content of the granules, and the appearance of the residues was consistent with the copresence in the granules of some organic material. Other mitochondrial structures were almost devoid of mineral. Thin sections of unincubated control mitochondria also were incinerated. They were found to contain appreciable amounts of intrinsic mineral, seemingly associated with membranes. The normal, dense matrix granules commonly seen in unaltered mitochondria could be seen in intact sections of these control preparations, but after burning no definite correspondence of any ash to the granules could be demonstrated. The normal granules perhaps do not contain mineral. Heating experiments on ash patterns of all the preparations demonstrated the thermal stability and crystallizability of the ash. The crystallized ash of the in vitro-produced dense granules was tentatively shown by electron diffraction to be beta-tricalcium phosphate (whitlockite). This, together with evidence from the literature, suggests that the original, noncrystalline mineral may be a colloidal, subcrystalline precursor of calcium-deficient hydroxyapatite. Experiments were performed on synthetic calcium phosphates for comparison. Other possible applications of the microincineration techniques are briefly discussed.

Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes.

Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.

Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria.

The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. The top fraction of the gradients contained a single polypeptide species (Mr approximately equal to 15,200) based upon SDS PAGE. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment (Mr approximately equal to 6,000) that has been purified by essentially the same procedures used for intact P16. The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods. Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. The labeling of P16 with [35S]methionine in primary rat hepatocyte cultures was inhibited by more than 90% by the cytoplasmic translation inhibitor cycloheximide, but unaffected by the mitochondrial-specific agent chloramphenicol. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function. Incubation of P16 with single strand phi X174 DNA, double strand (RF) phi X174 DNA, or Escherichia coli ribosomal RNA and subsequent analysis of the nucleic acid species for bound protein indicated a strong preference of P16 for single strand DNA and no detectable affinity for RNA or double strand DNA. Examination of P16-single strand phi X174 DNA complexes by direct electron microscopy revealed thickened, irregular fibers characteristic of protein-associated single strand DNA.

Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions.

The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product.

Biochemical and stereological analysis of rat liver mitochondria in different thyroid states.

The concentrations of the inner mitochondrial membrane markers cardiolipin and cytochrome alpha have been measured in liver homogenates and in purified mitochondria after thyroxine administration to thyroidectomized and normal rats. The biochemical results have been correlated with stereological electron micrographic analyses of hepatocytes in liver sections, and of isolated mitochondrial pellets. There were progressive and parallel increases in homogenate and mitochondrial cardiolipin concentration, and in mitochondrial cytochrome alpha concentration, after administration of 20 microgram of thyroxine on alternate days to thyroidectomized rats, and of 300 microgram on alternate days to normal rats. Electron microscope measurements showed marked differences in the shape of the mitochondria and in the number of cristae in different thyroid states. Hypothyroid mitochondria were shorter and wider than controls, and hyperthyroid mitochondria longer but of similar width. Mitochondrial volume per unit cell volume was virtually unchanged in hypo- and hyperthyroid animals. The most striking changes were a decrease in the area of the inner membrane plus cristae in thyroidectomized rats, and a substantial increase in membrane area after thyroxine administration. The biochemical and electron micrographic results indicate that, in rat liver, thyroid hormone administration leads to a selective increase in the relative amount of mitochondrial inner membranes, with little or no change in the mitochondrial volume per unit cell volume, or in total mitochondrial protein per unit total cell protein.

The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions.

Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-alpha-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.

Studies on the biochemistry of mitochondria and cell morphology in the neonatal swine hepatocyte.

Mitochondrial preparations isolated from neonatal swine hepatocytes show a marked increase in oxidative and concomitant phosphorylative capacity between birth and 2 days postpartum. There are no changes in the coupling parameters (respiratory control ratio and adenosine diphosphate/O ratio) with age. Changes in sedimentation properties in a sucrose gradient suggest qualitative changes in the mitochondria. Some of the lipid measurements (increased phospholipid) might be interpreted as supportive of this suggestion, although most could also be regarded as indicative of quantitative changes (increased number of mitochondria). Electron microscopy of isolated mitochondria and of the hepatocyte demonstrated an increased number of mitochondria but no change in shape, size, or structure as the pig developed. An increase in a number of cytoplasmic components (Golgi apparatus and endoplasmic reticulum) and a decrease in glycogen were also observed. The functional changes in mitochondria seem to occur within a short period of time (6-12 hr postpartum).

Subcellular morphometric and biochemical analysis 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.

Single strand-containing replicating molecules of circular mitochondrial DNA.

Mitochondrial DNAs (mtDNAs) from Chang rat solid hepatomas and Novikoff rat ascites hepatomas were examined in the electron microscope after preparation by the aqueous and by the formamide protein monolayer techniques. MtDNAs from both tumors were found to include double-forked circular molecules with a form and size suggesting they were replicative intermediates. These molecules were of two classes. In molecules of one class, all three segments were apparently totally double stranded. Molecules of the second class were distinguished by the fact that one of the segments spanning the region between the forks in which replication had occurred (the daughter segments) was either totally single stranded, or contained a single-stranded region associated with one of the forks. Daughter segments of both totally double-stranded and single strand-containing replicating molecules varied in length from about 3 to about 80% of the circular contour length of the molecule. Similar classes of replicating molecules were found in mtDNA from regenerating rat liver and chick embryos, indicating them to be normal intermediates in the replication of mtDNA All of the mtDNAs examined included partially single-stranded simple (nonforked) circular molecules. A possible scheme for the replication of mtDNA is presented, based on the different molecular forms observed

Optical diffraction studies of crystalline structures in electron micrographs. II. Crystalline inclusions in mitochondria of human hepatocytes.

Unit cell dimensions of mitochondrial crystals were determined by optical diffraction analysis of electron micrographs of human liver biopsy specimens. Identical unit cells were found in pathologic material obtained from six patients with Wilson's disease, from one patient with sickle-cell hepatitis, and from two normal subjects. These measurements led to the conclusion that the crystals observed in patients and in normal subjects were probably chemically identical. Furthermore, the relatively large size of the unit cell limits the choices for its constituents to phospholipid micelles or to relatively large protein molecules.

Cytochemistry of Golgi fractions prepared from rat liver.

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF(1), GF(2), GF(3)) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF(1) and GF(2), and along the outside of the cisternal membranes in GF(3). In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF(1) and within many VLDL-filled vacuoles in GF(1) and GF(2), indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF(3) and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF(1) and GF(2), and was not found in the cisternae in GF(3). The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF(1) and GF(3), representing primarily trans-Golgi elements from the secretory Golgi face, and GF(3) consisting largely of cis-Golgi components from the opposite face.

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria.

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 +/- 0.1, 0.6 mM NaHCO(3), had an electrophoretic mobility of - 2.88 +/- 0.01 micro/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 +/- 0.02, of rat liver mitoplasts was - 1.22 +/- 0.07, and of rat cerebral cortex mitoplasts - 0.91 +/- 0.04 micro/sec per v per cm. Treatment of these particles with 50 microg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in micro/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 microg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK(a) approximately 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.

Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matrix activities.

Treatment of the inner membrane matrix fraction of rat liver mitochondria with the nonionic detergent Lubrol WX solubilized about 70% of the total protein and 90% or more of the following matrix activities: malate dehydrogenase, glutamate dehydrogenase, and isocitrate dehydrogenase (NADP). The Lubrol-insoluble fraction was enriched in cytochromes, phospholipids, and a Mg(++)-stimulated ATPase activity. Less than 2% of the total mitochondrial activity of monoamine oxidase, an outer membrane marker, or adenylate kinase, an intracristal space marker could be detected in this inner membrane fraction. Electron micrographs of negatively stained preparations showed vesicles (

Vitamin A: concentration in the rat liver Golgi apparatus.

Vitamin A compounds (principally as retinyl esters) are concentrated in Golgi apparatus fractions from rat liver. The amounts vary with the vitamin A status of the liver and show an inverse relation to the concentration of ubiubinone. The results suggest a specific role of the Golgi apparatus in the mobilization or action, or both, of vitamin A compounds.

Mitochondrial autonomy: incorporation of monosaccharides into glycoprotein by isolated mitochondria.

Isolated intact mitochondria selectively incorporate monosaccharides from nucleotide diphosphate monosaccharides into protein. Fucose, mannose, glucose, and galactose were incorporated by the mitochondria into glycoprotein; xylose was not. Structural integrity of the mitochondria was not necessary for the incorporation of monosaccharide into glycoprotein; mitochondria broken by homogenization also incorporated monosaccharide. The monosaccharides incorporated into glycoprotein were localized in the inner mitochondrial membranes, the same membranes which contain the protein into which leucine is incorporated by the isolated mitochondria.

Mitochondrial thyroid hormone receptor: localization and physiological significance.

Binding studies of thyroid hormone to submitochondrial fractions from rat liver suggest that the component responsible for high-affinity, low-capacity (saturable) binding of hormones arises from the inner mitochondrial membrane. The partially purified component, approximately 150,000 daltons, appears to be half protein and half lipid, largely phospholipids, tentatively identified as lecithin, phosphatidyl ethanolamine, and cardiolipin. A similar hormone-binding macromolecule was found in mitochondria from rabbit kidney, from human liver and kidney, and from rat kidney, myocardium, skeletal muscle, intestinal mucosa, whole small intestine, adipose tissue, and lung. It was absent from mitochondria of adult rat brain, spleen, and testis, organs calorigenically unresponsive to thyroid hormones injected in vivo, but was present in mitochondria from brains of rats 12 days old and younger. The organ distribution of the hormone-binding protein and its presence in neonatal brain mitochondria supports the biological relevance of the mitochondrial component as a thyroid hormone receptor.

Specific lack of the hypermodified nucleoside, queuosine, in hepatoma mitochondrial aspartate transfer RNA and its possible biological significance.

Tumor nucleic acids have frequently been found to be deficient in methylated and other modified nucleotides. In particular, cytoplasmic transfer RNAs (tRNAs) from various neoplasms partially lack the hypermodified nucleoside queuosine, a modification specific for anticodons of histidine-, tyrosine-, asparagine-, and aspartic acid-accepting tRNAs. Using aspartate tRNA as an example, we show here that liver mitochondria contain tRNA fully modified with respect to queuosine, while the corresponding tRNA from mitochondria of Morris hepatoma 5123D completely lacks this constituent. The sequences of these tRNAs, which were determined by a highly sensitive 32P-postlabeling procedure entailing the direct identification of each position of the polynucleotide chains, were found to be (sequence in text) Lack of queuosine in the hepatoma mitochondrial tRNA may be due to the inavailability of queuine in the hepatoma mitochondria for incorporation into tRNA or to inhibition of the modifying enzyme, tRNA (guanine)-transglycosylase, in the tumor. Taking into account results of others indicating a possible involvement of the queuosine modification in differentiation of eukaryotic cells, we hypothesize that the queuosine defect may develop at an early stage of carcinogenesis (i.e., during the promotion phase) and be directly involved in abnormalities of mitochondria which have been observed frequently in transformed cells and tumors.

Role of lipid transfer proteins in the abnormal lipid content of Morris hepatoma mitochondria and microsomes.

The level of nonspecific lipid transfer protein in three Morris hepatomas of varying degrees of differentiation has been found to be less than 10% of the level in either host or control livers, whereas phosphatidylcholine transfer activity is slightly reduced for hepatomas compared to host livers (20 to 40%), though nearly the same as levels present in livers of non-tumor-bearing rats. Two major differences have been observed in the lipid compositions of tumor mitochondrial and microsomal membranes compared to those of normal membranes. For all three hepatomas, the cholesterol content of mitochondria is markedly elevated, and the phospholipid content of microsomes is reduced (per mg of protein). As a consequence of the above alterations, the cholesterol: phospholipid ratio of both the microsomal and mitochondrial membranes of these tumors is increased, and the extent of this increase correlates well with the degree of differentiation of the hepatomas. If an important physiological role of lipid transfer proteins is to transfer newly synthesized lipids from microsomes to other cellular organelles, one would expect a 10-fold decline of nonspecific lipid transfer protein activity in hepatomas to result in phospholipid and cholesterol accumulation in microsomes and depletion in mitochondria. The observed decline of phospholipid in microsomes and accumulation of cholesterol in mitochondria from hepatomas suggest that this is not an important function of the nonspecific lipid transfer protein. Finally, preliminary studies of the possibility that the elevated cholesterol: phospholipid levels in hepatoma membranes affect the activities of enzymes of these membranes are reported.

Further analysis of the type differences of rat-liver mitochondrial DNA.

1. The sequence of the small Hind III fragment F of rat-liver mitochondrial DNA (mtDNA) type A and type B was determined in order to investigate the nature of the differences between the two types of mtDNA and to determine its position in the Hind III fragment map. 2. The three differences found were point mutations. No deletion or insertion and no modification was observed. Two of the three differences affect the sequences which are recognition sites for Eco RI, Alu I and Taq I in type A, but not in type B, mtDNA. 3. The presence of an Eco RI restriction site only within the Hind III fragment F of type A shows that the fragment is situated in between the Hind III fragments A and E. 4. In one of the six reading frames, the Hind III fragment F contains the code for a carboxyl-terminal end of a polypeptide in which the three mutations do not lead to alterations in the possible aminoacid sequence. 5. The restriction sites for Taq I and a number of the sites for Alu I and Hae III were mapped. 6. The positions of the Hap II fragment J, and of a Hind III fragment G on the mtDNA were determined.

The internal concentration of K+ in isolated rat liver mitochondria.

A simple osmotic method has been developed to determine the internal K+ concentration of mitochondria by determining the concentration of external K+ at constant osmotic pressure at which metabolically inhibited mitochondria neither shrink nor swell. This concentration has been found to correspond to approx. 80-85 mM in freshly isolated mitochondria and considerably lower after additional centrifugation procedures. Since mitochondria are in osmotic equilibrium with the suspending medium (in this case, 0.32 osmolal), and K+ is the primary exchangeable internal ion, a significant proportion of the internal osmotic pressure must be exerted by the sucrose. Results for experiments determining internal K+ after centrifuging mitochondria at various G values confirm the reports of Sitaramam et al. (Sitaraman, V. and Sarma, M.K.J. (1981) Proc. Natl. Acad. Sci. USA 78, 3441-3445 and Sambasivarao, D. and Sitaramam, V. (1983) Biochim. Biophys. Acta 722, 256-270) that centrifugation induces the entry of sucrose in mitochondria isolated in a sucrose medium.