Search

The effect of 2 and 4 h of tourniquet ischaemia followed by 1 h of reperfusion on the major mitochondrial phospholipids and on the cytochrome c oxidase kinetic parameters has been investigated in rat skeletal muscle. There was no change either in the mitochondrial phospholipid content or in the Vmax and the Km of the enzyme after 2 h of ischaemia with and without subsequent reperfusion. Four hours of ischaemia had no effect on the lecithin and the cephalin content, while the cardiolipin content decreased as well as the Vmax of the enzyme (P less than 0.05). Tissue reperfusion caused a dramatic decrease in both cardiolipin (55% of the control, P less than 0.001) and Vmax (38% of the control, P less than 0.001). The corresponding reduction in lecithin and cephalin contents was 12% and 14% respectively (P less than 0.05). The Km remained unchanged at all conditions. These findings suggest that mitochondrial dysfunction in response to ischaemia and reperfusion could be a consequence of the reperfusion itself following severe ischaemia. The results are discussed in terms of cardiolipin peroxidation and cytochrome oxidase as a functional parameter.

A 43-year-old woman with progressive external ophthalmoplegia developed a bifascicular block and dilatation of the right ventricle during 4 years of follow-up. Histochemical and electron microscopy studies detected mitochondrial abnormalities in ocular, skeletal muscle and cardiac biopsies. This case registers disease progression from the external ocular to the skeletal and cardiac muscles. Mitochondrial DNA was deleted in relation to the morphological abnormality.

The immunocytochemical and immunochemical identification of beta-protein precursor in cultured vascular cells was undertaken using three types of antibodies to the synthetic predictive peptides (extracellular portion; 275-286, beta-protein portion; 597-620, C-terminal portion; 681-695) of beta-protein precursor. Monoclonal antibody directed toward the beta-protein portion stained the surface membrane of the cultured endothelial cells as well as cytoplasmic organelles. Immunoblotting of the subcellular fractions of the cell homogenate with three antibodies revealed that the plasma membrane associated beta-protein precursor consists of 105-130 kDa protein complexes and that the mitochondria-microsome-associated beta-protein consists of related 30-67 kDa protein complexes. The immunoreactivity of the protein bands was blocked by pretreating the monoclonal antibody with bovine serum albumin-conjugated beta-protein. These results indicate that the form of the beta-protein precursor or beta-protein-related components in cultured vascular cells is heterogeneous in terms of molecular size and subcellular localization.

We investigated the correlations of deletions of mitochondrial DNA in skeletal muscle with clinical manifestations of mitochondrial myopathies, a group of disorders defined either by biochemical abnormalities of mitochondria or by morphologic changes causing a ragged red appearance of the muscle fibers histochemically. We performed genomic Southern blot analysis of muscle mitochondrial DNA from 123 patients with different mitochondrial myopathies or encephalomyopathies. Deletions were found in the mitochondrial DNA of 32 patients, all of whom had progressive external ophthalmoplegia. Some patients had only ocular myopathy, whereas others had Kearns-Sayre syndrome, a multisystem disorder characterized by ophthalmoplegia, pigmentary retinopathy, heart block, and cerebellar ataxia. The deletions ranged in size from 1.3 to 7.6 kilobases and were mapped to different sites in the mitochondrial DNA, but an identical 4.9-kilobase deletion was found in the same location in 11 patients. Biochemical analysis showed decreased activities of NADH dehydrogenase, rotenone-sensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome c oxidase, four enzymes of the mitochondrial respiratory chain containing subunits encoded by mitochondrial DNA. We conclude that deletions of muscle mitochondrial DNA are associated with ophthalmoplegia and may result in impaired mitochondrial function. However, the precise relation between clinical and biochemical phenotypes and deletions remains to be defined.

E.p.r. (electron-paramagnetic-resonance) spectra of ubisemiquinone (QH) organic radicals and all of the known iron-sulphur centres were studied in normal and 'nickle-plated' pigeon heart mitochondria, submitochondrial particles and submitochondrial particles from which succinate dehydrogenase had been removed. Incubation of pigeon heart mitochondria, submitochondrial particles or succinate dehydrogenase-depleted submitochondrial particles with substrate in the presence of pure O2 results in the accumulation of Q-H. In mitochondria, the e.p.r. spectrum of Q-H is characterized by in-homogeneous line broadening. A heterogeneous population of semiquinones appears to be partly responsible for these effects in mitochondria. Additon of Ni(II) to mitochondria renders saturation of the Q-H resonance more difficult. On the other hand, the resonance in either submitochondrial particles or succinate dehydrogenase-depleted particles is narrower than the same spectrum in mitochondria, and saturates like a homogeneous line. The presence of Ni(II) in either of these preparations, further, has no effect on either the A-H spectrum or the saturation curve. Therefore QH appears to be situated on the exterior surface of the mitochondrion. Likewise, the e.p.r. spectra and saturation curves of iron-sulphur centre N-2 exhibit characteristics of inhomogeneous line broadening, not only in intact mitochondria but also in both submitochondrial particles and succinate dehydrogenase-depleted particles. Because of the small pool size of centre N-2, this effect is likely to arise from a spin interaction with some other component in the membrane. Ni(II) has no effect on the saturation in centre N-2 in mitochondria or submitochondrial particles, and only a marginal effect in the succinate dehydrogenase-depleted preparation. These results are indeterminate with respect to the position of centre N-2 in the membrane; but suggest that its distance from the succinate dehydrogenase binding site is on the order of 1 nm. All of the other ferredoxin-type iron-sulphur centres in both preparations were not affected by paramagnetic ions. Homogeneous e.p.r. spectra and saturation curves are observed for both of the HiPIP-type (high-potential iron-sulphur protein-type) iron-sulphur centres in mitochondrial centres S-3 and bc-3. Addition of No(II) to intact mitochondria results in a dipolar interaction with centre bc-3. No effect was observed on centre S-3 in either preparation. A comprehensive model is presented for the structure of the respiratory electron-transport system in mitochondria, based on e.p.r. relaxation studies in the present and the preceding paper. There is no direct evidence for transmembrane electron flow through any of the known energy-coupling sites in mitochondria, so that direct hydrogen atom transfer across the membrane (as a combination of H+ translocation coupled to electron flow) does not occur...

A 55-year-old man is presented who developed severe multifocal myoclonus and tonic clonic seizures in his early thirties, and progressive limb weakness in his mid forties, when a ragged red fibre myopathy was diagnosed. He went on to develop a distal motor neuropathy and respiratory failure. Respiratory function tests indicated respiratory failure secondary to respiratory muscle weakness and a central hypoventilation syndrome. CT scan revealed brain stem atrophy and brain stem evoked responses were abnormal. A sural nerve biopsy showed severe axonal degeneration. Cytochrome difference spectra and polarographic studies on isolated intact muscle mitochondria were normal. This study reports the association of respiratory failure and sleep apnoea with Fukuhara's syndrome and presents biochemical data suggesting that the mitochondrial respiratory chain may be intact in some patients with this syndrome.

Malic enzymes were studied in skeletal muscle from seven patients with Friedreich's ataxia (FA) and nine controls. Muscle contained three different malic enzymes. There were two strictly NADP+-dependent enzymes, one in the cytosol and one in mitochondria. These two enzymes are not allosteric. In FA muscle, activity of the mitochondrial NADP+-linked enzyme was significantly low and the cytosol NADP+-linked enzyme was significantly increased. A third malic enzyme, NAD+(P)-dependent, was found in the mitochondrial fraction. That enzyme had allosteric properties, and its activity was about the same in FA and control muscle.

We describe two subpopulations of actin antibodies isolated by affinity chromatography from a polyclonal antibody to chicken gizzard actin. One subpopulation recognizes gamma actins from smooth muscle and nonmuscle cells, but does not recognize alpha actin from skeletal muscle. The other subpopulation recognizes determinants that are common to alpha actin from skeletal muscle and the two gamma actin isotypes. Neither antibody recognizes cytoplasmic beta actin. Both antibodies recognize only actins or molecules with determinants that are also present in actins. By immunofluorescence we found that the anti-gamma actin colocalizes with mitochondria in fibers of mouse diaphragm, and that it does not bind detectably to the 1 bands of sarcomeres. The antibody that recognizes both alpha and gamma actins stains 1 bands intensely, as expected. We interpret these observations as preliminary evidence for selective association of gamma actin with skeletal muscle mitochondria and, more broadly, as evidence for subcellular sorting of isoactins.

The mitochondrial oxidative phosphorylation, calcium and magnesium contents, and swelling-contraction activity were investigated in relation to the progression of the hereditary hamster cardiomyopathy. The assessment was made in animals between 22 and 232 days of age, which were divided into 7 groups according to stage of disease. In 24-day-old hamsters prior to development of heart necrotic changes, the membrane permeability of isolated mitochondria was altered. In 50-day-old animals, at a stage of disease when myocardial cells undergo degeneration, a defect of oxidative phosphorylation resulting from an increase in mitochondrial calcium was demonstrated. With culmination of the heart necrotic changes, at close to 100 days of age, mitochondrial dysfunction and calcium overload were maximal. There was a transient improvement during the healing stage, but the situation deteriorated with the occurrence of circulatory failure. Since the mitochondrial respiratory pattern and calcium overload parallel the cardiac degeneration, it is inferred that the cell energy depletion is a functional consequence of an abnormal calcium influx.

E.p.r.(electron-paramagnetic-resonance) spectra of the ferricytochromes were studied in normal and 'nickel-plated' pigeon heart mitochondria and pigeon heart submitochondrial particles. NiCL2 added to either mitochondria or particles was bound completely to the membranes, but none was transported across the vesicles. Hence, any perturbations of the haem e.p.r. spectra by Ni(II) should occur only for those cytochromes in close proximity to the exterior surface. Whenever Ni(II) can approach to within 1 nm of cytochrome haem. the consequent acceleration of the haem e.p.r. relaxation kinetics should elicit dipolar line broadening. Relaxation acceleration should also increase the incident power level required to saturate the haem e.p.r. signal. In pigeon heart mitochondria, at least three e.p.r. resonances, attributable in part to cytochromes c1, bK and br, are observed at gz=3.3 resonance. In these submitochondrial particles, the peak at gz=3.5 is missing, and the resonance at gz=3.6 resolves into two components, neither of which is sensitive to added Ni(ii). Addition of free haemin (ferric, a paramagnetic anion) to intact mitochondria elicits the same e.p.r. signal changes as does a preparation of submitochondrial particles. Saturation curves for cytochrome oxidase obtained for e.p.r. spectra of the high-spin form (g = 6) and the low-spin form (gz=3.1) also reveal no effect of Ni(II) on the haem e.p.r. relaxation in either mitochondria or inverted submitochondrial particles. Further, Ni(II) fails to alter the spectra or saturation properties of cytochrome c in either mitochondria or submitochondrial particles therefrom. Only with a 50-fold molar excess of Ni(II) can one accelerate the e.p.r. relaxation of cytochrome c in aqueous solution, although other more subtle types of magnetic interactions may occur between the cytochrome and either Ni(II) or ferricyanide. Addition of haemin to mitochondria likewise failed to alter the e.p.r. characteristics of either cytochrome c or cytochrome oxidase. The present observations strongly suggest that cytochromes bK, br and c1 reside on the exterior surface of the inner mitochondrial membrane. On the other hand, we find no positive evidence for the location of cytochrome c or cytochrome oxidase haem groups within 1 nm of either membrane surface. Because of possible shielding effects from the protein moieties, however, we cannot unequivocally assign the location of the haem groups to the membrane interior. The present results are not inconsistent with the observations of other investigators who used different techniques. However, it is clear that any model of energy coupling in mitochondrial oxidative phosphorylation must account for the positioning of all the b-c cytochrome haem groups on the outside.

Mitochondrial calcium overloading was investigated in the genetically dystrophic mouse (strains 129/ReJ dy/dy) as a possible contributing factor to the development of muscle fiber necrosis. Mitochondrial calcium concentrations were significantly elevated in both skeletal muscle and heart organelles. Because mitochondria were isolated in the presence of ruthenium red this finding was not the result of an artefact of isolation. State 3 respiration rates and concomitantly the respiratory control ratios were slightly decreased in skeletal muscle, but not in heart mitochondria. This abnormality could result from calcium overloading in a small fraction of the mitochondria. Fractionation of skeletal muscle mitochondria on sucrose gradients gave two distinct populations of dystrophic organelles, one with high calcium, whereas normal skeletal muscle mitochondria and heart organelles showed only one broad band on the gradient. The results support the idea that both skeletal muscle and heart are affected in dystrophic mice, strain 129/ReJ dy/dy and also that in the dystrophic mouse the process of cell necrosis is associated with cellular calcium overloading.

Serial sections of longissimus dorsi and rectus femoris muscles from 15 Yorkshire breed pigs (live weights 24-46 and 49-139 kg) were stained for glycogen (PAS) and a mitochondrial enzyme (NAD tetrazolium reductase). Muscle fibres with a low mitochondrial content in both muscles were more frequently PAS-positive than fibres with a high or intermediate mitochondrial content. However, some pigs had all their muscle fibres PAS-positive while one pig with a high post-mortem muscle pH had all rectus femoris fibres PAS-negative. Relative to lighter weight pigs, longissimus dorsi muscles of heavy pigs tended to have less fibres with a high mitochondrial content and less fibres with a positive PAS reaction. Compared to longissimus dorsi muscles, rectus femoris muscles had more fibres with a high mitochondrial content and less with a positive PAS reaction. All fibres in both muscles became PAS-negative with an accompanying decrease in pH by 24 hr post-mortem. Fibres from longissimus dorsi muscles frequently had PAS-positive sarcoplasmic cores between their myofibrils. Heavy pigs tended to have larger cores (up to a mean maximum diameter of 13.4 mum), more fibres with cores, and more cores per fibre. The pigs involved exhibited no other ante- or post-mortem muscle abnormalities.

Quinacrine (Atebrin), an antimalarial drug that binds to adenine nucleotides, was used to label paraneurons. The drug was dissolved in physiological saline and intraperitoneally injected (rats, mice, guinea pigs) in concentrations of 120 or 200 mg/kg body weight. The animals were killed three days later and paraneurons were studied in the fluorescence microscope aftre freeze drying. Bright fluorescence is observed in adrenomedullary cells, SIF cells, carotid body chief cells, pancreatic islet cells, anterior pituitary cells, pinealocytes, and mast cells. Weak fluorescence occurs in thyroid parafollicular Merkel cells, and a few entero-endocrine cells. It is suggested that quinacrine binds to ATP or related adenine nucleotides which are stored in secretory granules of paraneurons. Varying fluorescence intensity seems to depend on different concentrations of adenine nucleotides within the storage granules, as well as on the different size and number of these granules in various paraneurons. Besides formaldehyde-induced fluorescence of biogenic amines, HCl-basic dye staining methods and electron microscopy, the method is useful to identify and define paraneurons.

Biochemical studies have been performed in muscle, liver, leukocytes, and fibroblasts from patients suffering from the Zellweger syndrome. Oxidation rates of [1-14C]pyruvate, [U-14C]malate, and [1-14C]2-oxoglutarate were strongly reduced in skeletal muscle homogenate. Oxygen consumption in isolated skeletal muscle mitochondria could only be stimulated by ADP in the presence of ascorbate and N,N,N1,N1-tetramethyl-p-phenylenediamine. Cytochrome contents in heart muscle and liver mitochondria were normal. A very low activity of succinate-ubiquinone oxidoreductase was found in liver homogenate of two patients. From the effect of 2-thenoyltrifluoroacetone on the succinate-phenazine methosulphate oxidoreductase activity, a nearly competitive inhibition with respect to phenazine methosulphate was demonstrated in contrast with a non-competitive inhibition in controls. Normal oxidation rate of [1-14C]pyruvate and [2-14C]pyruvate was found in leucocytes and fibroblasts. Lactate and pyruvate levels were normal in serum and cerebrospinal fluid and beta-hydroxybutyrate and acetoacetate levels were normal in blood. The ratios lactate/pyruvate and beta-hydroxybutyrate/acetoacetate were normal as well. These findings point to a defect in the electron transport chain at the succinate-ubiquinone oxidoreductase level. This defect might be related to the absence of peroxisomes in the cells of Zellweger patients.

Electron-microscopic cytochemical studies on satellite cells of normal human skeletal muscle were carried out using the concanavalin A-peroxidase (Con A-HRP) coupling method. Con A-binding sites, which probably correspond to glycoproteins, were found to be associated with the cell surface, smooth surfaced vesicles, nuclear envelope and endoplasmic reticulum of the satellite cells and were also identified at the cell surface of the adjacent muscle fiber. The possible relationships of these observations to the functions of satellite cells are discussed.

Abnormalities have been noticed in the phospholipid and cholesterol composition of the atrophied gastrocnemius muscle of frog denervated for 1 month. Cholesterol : phospholipid molar ratios in the muscle increased on denervation. Sphingomyelin and cardiolipin fractions increased in contrast to phosphatidyl choline, phosphatidyl serine and phosphatidyl ethanolamine in the sarcoplasmic reticulum (SR) of denervated muscle. Na-azide sensitive Ca2+ ATPase activity of the mitochondria did not alter whereas that of SR decreased on denervation. Phospholipase C digestion impaired the organelle Ca2+-ATPase activity. The above abnormalities in enzyme activities have been correlated to the changes in the lipid composition of the denervated muscle. On the basis of these changes it is discussed that the primary change in the muscle due to denervation is the change in the permeability of the membrane.

The male rabbit's external urethral sphincter was investigated with O'Farrell's 2-dimensional electrophoretic analysis of myosin light chains and electron microscopy. Its pattern of myosin light chains was different from that of the soleus (predominantly slow twitch muscle) but was very similar to that of the psoas (predominantly fast twitch muscle). Ultrastructurally it was shown to be red muscle resembling the soleus. Therefore the fiber type of the rabbit's external urethral sphincter was determined to be the red (fast) type.

1. High efficiency of oxidative phosphorylation and a good respiratory control in liver, heart and somatic muscle mitochondria of the lamprey (Lampetra fluviatilis) were observed when the particles were isolated in a complex sucrose medium containing EDTA, heparin and nicotinamide. The coupling properties of these mitochondria were further improved by including serum albumin in the incubation medium. 2. The content of total adenine nucleotides in lamprey mitochondria was between 4 and 6 nmoles/mg protein. The translocation of these nucleotides across mitochondrial membrane was stimulated by serum albumin. 3. Lamprey mitochondrial phospholipids contain a large proportion (64-72%) of polyunsaturated fatty acids. 4. Electron micrographs of mitochondria from lamprey liver, heart and somatic muscle are presented.

Report on a sporadic case with congenital slowly progressive neuromuscular disease. Light microscopic, ultrastructural and histochemical changes of muscle biopsy reveal the characteristics of central core disease with "structured cores" in type II-fibers.

In Rhesus monkeys, nicotinic acid given daily by subcutaneous injection for 5 weeks brought about a reversible decrease in total cholesterol concentration in skeletal muscle and skin to about half the normal value. The decrease in cholesterol concentration was due to a net loss of cholesterol from muscle, since the treatment had no effect on the water content or on the percentage of DNA or protein in fresh tissue. In muscle, free cholesterol was lost in preference to esterified cholesteol, but in skin both cholesterol fraction were affected to about the same extent. Analysis of the cholesterol content of subcellular fractions of homogenates of muscle showed that loss of cholesterol occurred mainly from the soluble fraction and the 800-g sediment, with no significant loss from the mitochondrial or microsomal fractions.

Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N2-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600 A-900 A in diameter in the normal myocardium of the sand rat and the mouse and from 250 A-400 A in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.

A microsomal preparation with a high ability for Ca2+ uptake has been isolated from pigeon heart. A method of further purification of Ca2+-accumulating system of heart, based on the ability of sarcoplasmic reticulum for the energy-dependent Ca2+ accumulation in the presence of oxalate, has been developed. Upon centrifugation in the gradient of sucrose and KCl concentration the fragments of sarcoplasmic reticulum, rendered "heavy" by calcium oxalate, can be separated from foreign cell membranes. The main component of heart "calcium pump" is Ca2+-dependent ATPase (making up to about 50% of all proteins of the purified reticulum), having a molecular weight of 100.000--105.000. Specific activity of heart Ca2+-ATPase as well as the ability of purified heart sarcoplasmic reticulum for Ca2+ uptake are only slightly less than those of the skeletal muscle reticulum. The data obtained suggest that heart sarcoplasmic reticulum may be efficient for providing heart muscle relaxation.

Using a DW 2 dual wavelength spectrophotometer (AMINCO) and murexide as a Ca2+ sensitive indicator it is shown that guinea-pig heart mitochondria can release accumulated Ca2+ without the influence of non-physiological material. The main factor which decides if accumulated Ca2+ is spontaneously released is the Ca2+/mitochondrial-protein ratio. Under appropriate assay conditions a critical intramitochondrial Ca2+ concentration is reached. Increasing this concentration leads to Ca2+-release.

Our data clearly demonstrate that protective effect of phosphate and protective effect of mersalyl against NEM-inhibition of phosphate transport act at the level of two kinds of proteins. (1)Two major components are phosphate and nigericin NEM sensitive. According to our previous data [13] it has been also demonstrated that these two proteins components are valinomycin NEM sensitive (results not shown here) suggesting a relationship between these proteins and the energy linked proton translocation process. Relationships between these proteins and the phosphate translocation process are not evident and are under further investigations. (2) Two other insoluble major components localised at the level of the subparticular fraction are mersalyl NEM sensitive. We can suggest that these proteins are implicated in the translocation of phosphate in pig heart mitochondria.

Tissue sections of both transiently and persistently ischemic musculus latissimus dorsi of rabbits show degenerated and necrotic cells in varying degrees. Degenerated mitochondria appearing in the cells contain moderate electron-dense fluffy intramatrical deposits. However, only in the transient ischemia sample can other two kinds of electron-dense granules be seen between myofibrils. According to their ultrastructure, size and distribution, the larger granules are considered the degenerated mitochondria, and the smaller ones the degenerated sarcoplasmic reticulum. X-ray microanalysis proves that these two kinds of granules contain high concentration of calcium, while the fluffy deposits contain little calcium. The findings suggest that the calcium deposits in the degenerated and necrotic cell seen under light microscope occur in mitochondria and sarcoplasmic reticulum of the transiently ischemic muscle cell. The conditions of calcium accumulation in ischemic muscle cells are discussed.

Glycerol-3-phosphate dehydrogenase (E. C. 1. 1. 99. 5) was solubilized from rabbit skeletal muscle mitochondria by Triton X-100 and purified through hydroxyapatite column chromatography, DEAE-Sepharose CL-6B column chromatography and sucrose density gradient ultracentrifugation. The preparation was electrophoretically pure, the total recovery was 10% and the specific activity had been increased 200-fold. The apparent molecular weight of the enzyme polypeptide was 69,000, it existed in the form of enzyme-Triton X-100 complex with a Stokes' radius of 59 A and a sedimentation coefficient of 10.7 S. There were 1.7 mg Triton X-100 and 26 micrograms phospholipid per mg protein of the preparation. The enzyme absorbed at 410 and 460 nm which could be attributed to non-haem iron and FAD respectively. Both of the absorption would be largely diminished by adding the substrate glycerol-3-phosphate.

We determined the nucleotide sequences of junctional regions associated with large deletions of mitochondrial DNA found in four unrelated individuals with a phenotype of chronic progressive external ophthalmoplegia. In each patient, the deletion breakpoint occurred within a directly repeated sequence of 13-18 base pairs, present in different regions of the normal mitochondrial genome-separated by 4.5-7.7 kilobases. In two patients, the deletions were identical. When all four repeated sequences are compared, a consensus sequence of 11 nucleotides emerges, similar to putative recombination signals, suggesting the involvement of a recombinational event. Partially deleted and normal mitochondrial DNAs were found in all tissues examined, but in very different proportions, indicating that these mutations originated before the primary cell layers diverged.

The peptides of purified complex III from beef heart mitochondria have been studied by electrophoresis on dodecylsulfate gels. Of the 12 peptides consistently observed, only eight appear to be integral peptides of the functional complex. Attempts to identify these peptides have been made through co-electrophoresis of complex III and fractions in which the individual peptides were either purified or greatly enriched. Electrophoresis of complex III preparations which were not reduced by mercaptoethanol indicates that intermolecular disulfide bonds play no significant role in stabilizing the complex.

Increases in oxidative capacity of skeletal muscle which has undergone training is well documented. The concomitant shifts in mitochondrial size and cytochrome content have varied with different investigations. In this study the shifts in oxidative capacity, SDH activity, thiolase activity, was measured in two sizes of heart and skeletal muscle mitochondria of rats undergoing 4, 8, and 16 weeks of training. In addition cytochrome content was measured in both sizes of mitochondria of the skeletal muscle. The small mitochondria of skeletal muscle showed the significant gains in number during the first 8 weeks of training while the large mitochondria increased in number during the last 8 weeks of training. The oxygen consumption, SDH activity, thiolase activity and cytochrome content all showed varying peaks in activity and content in the different sizes of mitochondria over the 16 weeks of training. In evaluating training regimes' effects on skeletal muscle it appears important to take into consideration the shifts in size of mitochondria as well as oxidative capacity and enzymatic activity alterations. There were no alterations observed in mitochondrial size in cardiac tissue.