Cytoplasm-to-myonucleus ratios and succinate dehydrogenase activities in adult rat slow and fast muscle fibers

The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n = 54; 9 ± 3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabelled with fast and slow myosin heavy chain monoclonal antibodies. Mean ± S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112 ± 69 vs. 34 ± 21 x 10 ³µm ³) than fast and slow soleus fibers (40 ± 20 vs. 30 ± 14 x 10 ³µm ³), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (<70 µm) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (>70 µm) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116 ± 51 vs. 55 ± 22 and 44 ± 23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.     

Variation and limitations in fiber enzymatic and size responses in hypertrophied muscle.

The present study was designed to determine whether the degree and kind of adaptation of a muscle fiber to a functional overload (FO) are determined by properties that are intrinsic to that fiber. The study also addresses the question of the capability of fibers to maintain a normal level of coordination of proteins per fiber as fiber volume changes dramatically. The plantaris muscle of six adult female cats was overloaded for 12 wk by bilateral synergist removal. Plantaris muscle fiber mean size doubled after FO, although some very small fibers that stained dark for adenosinetriphosphatase (ATPase) were observed in some of the FO muscles. There appeared to be no change in total succinate dehydrogenase activity per fiber. A reduction in succinate dehydrogenase activity per unit volume was observed in a substantial number of fibers, reflecting a disproportionate increase in fiber volume relative to mitochondrial volume. In contrast, total alpha-glycerophosphate dehydrogenase activity and actomyosin ATPase activity increased as fiber size increased, whereas there was no change in alpha-glycerophosphate dehydrogenase and ATPase activities per unit volume. Control and FO muscle fibers generally expressed either a fast or slow myosin heavy chain type, but in some cases FO muscle fibers expressed both fast and slow myosin heavy chains. The persistence of variability in fiber sizes and enzyme activities in fibers of overloaded muscles suggests a wide range in the adaptive potential of individual fibers to FO. These data indicate that a severalfold increase in cell size may occur without significant qualitative changes in the coordination of protein regulation associated with metabolic pathways and ATP utilization.     

Quantitative succinate dehydrogenase analysis in normal and ragged-red muscle fibers

Single fiber analyses were performed in normal and diseased muscle by means of a high-resolution microphotometric method. We investigated the activity distribution of a mitochondrial marker enzyme, succinate dehydrogenase, within single muscle fibers. We differentiated between the central and the subsarcolemmal region. Both normal muscle fibers, and ragged-red fibers from patients with a mitochondrial myopathy showed significantly higher succinate dehydrogenase activities in the subsarcolemmal region. Since the fibers' supply of oxygen is accomplished by diffusion from capillaries located close to the sarcoplasmic membrane our results are of functional importance.     

Quantitative succinate dehydrogenase analysis in normal and ragged-red muscle fibers

Summary Single fiber analyses were performed in normal and diseased muscle by means of a high-resolution microphotometric method. We investigated the activity distribution of a mitochondrial marker enzyme, succinate dehydrogenase, within single muscle fibers. We differentiated between the central and the subsarcolemmal region. Both normal muscle fibers, and ragged-red fibers from patients with a mitochondrial myopathy showed significantly higher succinate dehydrogenase activities in the subsarcolemmal region. Since the fibers' supply of oxygen is accomplished by diffusion from capillaries located close to the sarcoplasmic membrane our results are of functional importance.     

Fibre-optic spectrophotometry of immature bovine skeletal muscles and the cellular distribution of myoglobin and succinate dehydrogenase

Summary Samples of diaphragm and pectoralis profundus were taken from nine calves with a range of blood haemoglobin levels of 4 to 8.5 g/100 ml. In both muscles, fibres with strong succinate dehydrogenase activity contained myoglobin, but in the pectoralis there were many fibres with strong alkaline ATPase activity and weak succinate dehydrogenase activity that had low or undetected levels of myoglobin. The whole cross-sectional area of individual fibres was scanned to map the distribution of succinate dehydrogenase activity. Among fibres with similar levels of ATPase activity, those from the diaphragm had greater succinate dehydrogenase activity than those from the pectoralis. Subsarcolemmal succinate dehydrogenase activity was greater than the axial succinate dehydrogenase activity, and radial gradients of succinate dehydrogenase activity were steepest in the diaphragm. For pectoralis fibres with weak ATPase, the mean and the axial succinate dehydrogenase activities were correlated with blood haemoglobin levels (r=0.62 and r=0.61, respectively;P<0.05 with a Student'st-test). Muscle colour was measured directly by fibre-optic spectrophotometry and correlations of absorbance with succinate dehydrogenase activity were obtained. Absorbance at 620 nm 24 h post-mortem was correlated with succinate dehydrogenase activity in pectoralis fibres with weak ATPase (r=0.81;P<0.005).     

An antimycin-insensitive succinate-cytochrome c reductase activity in pure reconstitutively active succinate dehydrogenase

Antimycin-insensitive succinate-cytochrome c reductase activity has been detected in pure, reconstitutively active succinate dehydrogenase. The enzyme catalyzes electron transfer from succinate to cytochrome c at a rate of 0.7 mumole succinate oxidized per min per mg protein, in the presence of 100 microM cytochrome c. This activity, which is about 2% of that of reconstitutive (the ability of succinate dehydrogenase to reconstitute with coenzyme ubiquinone-binding proteins (QPs) to form succinate-ubiquinone reductase) or succinate-phenazine methosulfate activity in the preparation, differs from antimycin-insensitive succinate-cytochrome c reductase activity detected in submitochondrial particles or isolated succinate-cytochrome c reductase. The Km for cytochrome c for the former is too high to be measured. The Km for the latter is about 4.4 microM, similar to that of antimycin-sensitive succinate-cytochrome c activity in isolated succinate-cytochrome c reductase, suggesting that antimycin-insensitive succinate-cytochrome c activity of succinate-cytochrome c reductase probably results from incomplete inhibition by antimycin. Like reconstitutive activity of succinate dehydrogenase, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase is sensitive to oxygen; the half-life is about 20 min at 0 degrees C at a protein concentration of 23 mg/ml. In the presence of QPs, the antimycin-insensitive succinate-cytochrome c activity of succinate dehydrogenase disappears and at the same time a thenoyltrifluoroacetone-sensitive succinate-ubiquinone reductase activity appears. This suggests that antimycin-insensitive succinate-cytochrome c reductase activity of succinate dehydrogenase appears when succinate dehydrogenase is detached from the membrane or from QPs. Reconstitutively active succinate dehydrogenase oxidizes succinate using succinylated cytochrome c as electron acceptor, suggesting that a low potential intermediate (radical) may be involved. This suggestion is confirmed by the detection of an unknown radical by spin trapping techniques. When a spin trap, alpha-phenyl-N-tert-butylnitrone (PBN), is added to a succinate oxidizing system containing reconstitutively active succinate dehydrogenase, a PBN spin adduct is generated. Although this PBN spin adduct is identical to that generated by xanthine oxidase, indicating that a perhydroxy radical might be involved, the insensitivity of this antimycin-insensitive succinate-cytochrome c reductase activity to superoxide dismutase and oxygen questions the nature of this observed radical.     

Tumor necrosis factor-α in macrophages of heart,liver, kidney,and in the pituitary gland

The relationship between myonuclear number, cellular size, succinate dehydrogenase activity, and myosin type was examined in single fiber segments (n=54; 9±3 mm long) mechanically dissected from soleus and plantaris muscles of adult rats. One end of each fiber segment was stained for DNA before quantitative photometric analysis of succinate dehydrogenase activity; the other end was double immunolabelled with fast and slow myosin heavy chain monoclonal antibodies. Mean±S.D. cytoplasmic volume/myonucleus ratio was higher in fast and slow plantaris fibers (112±69 vs. 34±21x10³ m³) than fast and slow soleus fibers (40±20 vs. 30±14x10³ m³), respectively. Slow fibers always had small volumes/myonucleus, regardless of fiber diameter, succinate dehydrogenase activity, or muscle of origin. In contrast, smaller diameter (<70 m) fast soleus and plantaris fibers with high succinate dehydrogenase activity appeared to have low volumes/myonucleus while larger diameter (>70 m) fast fibers with low succinate dehydrogenase activity always had large volume/myonucleus. Slow soleus fibers had significantly greater numbers of myonuclei/mm than did either fast soleus or fast plantaris fibers (116±51 vs. 55±22 and 44±23), respectively. These data suggest that the myonuclear domain is more limited in slow than fast fibers and in the fibers with a high, compared to a low, oxidative metabolic capability.     

Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity

Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.     

The participation of primary amino groups of succinate dehydrogenase in the formation of succinate-Q reductase

(1) Purified succinate dehydrogenase contains about 49 mol of lysine residues per mol enzyme. Titration of succinate dehydrogenase with fluorescamine indicates that half the lysyl groups are located on the surface of the protein and the other half are buried inside. (2) The reconstitutive activity and the low Km ferricyanide reductase activity of succinate dehydrogenase decreased as the extent of alkylation of amino groups by fluorescamine increased. (3) The inhibitory effects of fluorescamine on both activities are parallel and are succinate concentration dependent. (4) Alkylation of the native succinate-Q reductase by fluorescamine does not affect the enzymatic activity or alter the enzyme kinetic parameters. This indicates that the inhibitory effect of fluorescamine on succinate dehydrogenase is due to the modification of a specific amino group(s) on succinate dehydrogenase which is essential in the interaction with QPs to form succinate-Q reductase. The participation of an ionic group in the formation of succinate-Q reductase supports the idea of the involvement of ionic interaction between succinate dehydrogenase and QPs.     

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.     

Soluble succinate dehydrogenase from the halophilic archaebacterium, Halobacterium halobium

Succinate dehydrogenase activity was found in both the cytoplasmic and the membrane fractions from disrupted Halobacterium halobium cells. The cytoplasmic enzyme was found to be soluble in aqueous media and had an apparent molecular weight of 90,000. The enzyme activity of the cytoplasmic succinate dehydrogenase was salt dependent, with preference for KCl over KNO3. The Km values for succinate of the soluble and the membrane-bound succinate dehydrogenases from H. halobium were 2.3 +/- 0.3 and 0.7 +/- 0.1 mM, respectively. The soluble succinate dehydrogenase was obtained from two different strains of H. halobium and was obtained independently of the method used to disrupt the bacteria. Thus, the archaebacterium, H. halobium, contains a succinate dehydrogenase which differs from the succinate dehydrogenase in most eucaryotic and eubacterial cells, where the enzyme is tightly membrane-bound.     

Metabolic alterations in skeletal muscle of chronically streptozotocin-diabetic rats

This study was accomplished to determine the effects of chronic streptozotocin diabetes and insulin treatment on selected enzymes and substrates used in energy transduction in muscles composed of different muscle fiber types. Triglyceride concentration in all the muscles of diabetic rats was significantly elevated. Glycogen and protein concentrations were unchanged. The enzyme activities of hexokinase and alanine aminotransferase were significantly reduced and 3-hydroxyacyl-CoA dehydrogenase increased in all the muscles. Declines in phosphofructokinase, lactate dehydrogenase, citrate synthase, and succinate dehydrogenase activities were found in the red gastrocnemius and plantaris. Glycerol-3-phosphate dehydrogenase activity was lower than normal in the red gastrocnemius. Insulin treatment to the diabetic rats returned the altered triglyceride content and enzyme activities to normal, with exception of the lower alanine aminotransferase activity in the red gastrocnemius and plantaris. However, this enzyme was significantly ameliorated when compared with the untreated diabetic rats. The findings show that hypoinsulinism has a differential effect on the enzymatic profile of the different skeletal muscle fiber types, with those of the red gastrocnemius being most severely affected. Insulin treatment returned the enzymatic profile of the fiber types in diabetic rats to essentially normal.     

Histochemical and electronmicroscopical analysis of muscle fiber in myotomes of teleost fish (Noemacheilus barbatulus L.)

A quantitative histochemical study was carried out on axial musculature of Noemacheilus barbatulus L. On the basis of succinate dehydrogenase (SDH) and myofibrillar ATP-ase activity, 5 types of muscle fibers are described. When the SDH method was used, red, tonic, intermediate, and white muscle fibers were easily observed. However, histochemical reaction for myofibrillar ATP-ase activity, after alkaline preincubation (pH = 10.4), revealed another type of fiber zone laying between the intermediate and white muscle fiber regions and forming a transitional zone. Electron microscopic observation showed significant differences in sarcomere organization and thickness of myosin filaments of the various muscle fiber types.     

Reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones.

The succinate dehydrogenase isolated from Bacillus subtilis was found to catalyze the oxidation of succinate with hydrophilic quinones. Either naphthoquinones or benzoquinones served as acceptors. The enzyme activity increased with the redox potential of the quinone. The highest turnover number was commensurate with that of the bacterial succinate respiration in vivo. The succinate dehydrogenase was similarly active in fumarate reduction with quinols. The highest activity was obtained with the most electronegative quinol. The fumarate reductase isolated from Wolinella succinogenes catalyzed succinate oxidation with quinones and fumarate reduction with the corresponding quinols at activities similar to those of the B. subtilis enzyme. Succinate oxidation by the lipophilic quinones, ubiquinone or vitamin K-1, was monitored as cytochrome c reduction using proteoliposomes containing succinate dehydrogenase together with the cytochrome bc1 complex. The activity with ubiquinone or vitamin K-1 was commensurate with the succinate respiratory activity of bacteria or of the bacterial membrane fraction. The results suggest that menaquinone is involved in the succinate respiration of B. subtilis, although its redox potential is unfavorable.     

Lysis of bacterial protoplasts and spheroplasts and suppression of their dehydrogenase activity by neotehomycin]

Neotelomycin induced lysis of the protoplasts of Bac. megaterium and inhibited their succinate dehydrogenase activity. Direct correlation between the lytic activity of the antibiotic and its effect on succinate dehydrogenase was found. Neotelomycin had no effect on the dehydrogenase activity of the protoplast lysates. Possibly, suppression of the protoplast succinate dehydrogenase of Bac. megaterium under the effect of neotelomycin was due to significant structural changes caused by the antibiotic in the protoplast membranes and leading to their lysis and not to the direct effect on the enzyme. Neotelomycin had practically no effect on the spheroplast dehydrogenase activity of E. coli resistant to the antibiotic and did not induce their lysis. Resistance of E. coli to neotelomycin must be associated not with the presence of the antibiotic non-permeable cell wall but the peculiar properties of the membrane cytoplasm.     

A correlation of the intensity of the gas discharge luminescence of the skin and the activity of succinate dehydrogenase in blood lymphocytes in different states of the body

The integral area of gas discharge luminescence of the hand finger skin and the activity of succinate dehydrogenase in lymphocytes in blood smears from patients with food allergia and attendant hypertension has been measured. Succinate dehydrogenase in these patients was hyperactivated or inhibited as compared with healthy persons. At more substantial deviations of activity, a more clearly pronounced hypertension was observed. The area of luminescence of the skin in some patients was near the upper boundary of norm or was beyond its limits. A high degree of correlation (r = 0.85) between the two parameters has been revealed. At moderate and high succinate dehydrogenase activity, the parameters of gas-discharge visualization decreased; the inhibition of succinate dehydrogenase activity enhances, the gas-discharge visualization increases. This probably indicates the contribution to the irradiation from hand fingers of the superoxide formed in the respiratory chain of mitochondria in the region of coenzyme Q upon its incomplete reduction caused by the inhibition of succinate dehydrogenase.     

Pyridoxal phosphate-induced dissociation of the succinate: ubiquinone reductase

Treatment of the soluble ubiquinone-deficient succinate: ubiquinone reductase with pyridoxal phosphate results in the inhibition of the carboxin-sensitive ubiquinone-reductase activity of the enzyme. The inactivation is prevented by the soluble homolog of ubiquinone (Q2) but is insensitive to the dicarboxylates interacting with the substrate binding site of succinate dehydrogenase. The reactivity of the pyridoxal phosphate-inhibited enzyme with different electron acceptors suggests that the observed inhibition is due to the dissociation of succinate dehydrogenase from the enzyme complex. The soluble succinate dehydrogenase was recovered in the supernatant after treatment of the insoluble succinate: ubiquinone reductase with pyridoxal phosphate. The data obtained strongly suggest the participation of amino groups in the interaction between succinate dehydrogenase and the ubiquinone reactivity conferring peptide within the complex.     

Interaction of succinate dehydrogenase with succinate and oxaloacetate during enzyme activation-deactivation

The interaction of succinate dehydrogenase from the bovine adrenal cortex with succinate and oxaloacetate was studied in the process of its activation-deactivation. It is supposed that an intermediate unstable complex of succinate dehydrogenase with oxaloacetate plays an important role in the changed enzymic activity.     

Decreased Succinate Dehydrogenase Activity of Gamma and Alpha Motoneurons in Mouse Spinal Cords Following 13 Weeks of Exposure to Microgravity

Cell body size and succinate dehydrogenase activity of motoneurons in the dorsolateral region of the ventral horn in the lumbar and cervical segments of the mouse spinal cord were assessed after long-term exposure to microgravity and compared with those of ground-based controls. Mice were housed in a mouse drawer system on the International Space Station for 13 weeks. The mice were transported to the International Space Station by the Space Shuttle Discovery and returned to Earth by the Space Shuttle Atlantis. No changes in the cell body size of motoneurons were observed in either segment after exposure to microgravity, but succinate dehydrogenase activity of small-sized (<300 μm²) gamma and medium-sized (300–700 μm²) alpha motoneurons, which have higher succinate dehydrogenase activity than large-sized (>700 μm²) alpha motoneurons, in both segments was lower than that of ground-based controls. We concluded that exposure to microgravity for longer than 3 months induced decreased succinate dehydrogenase activity of both gamma and slow-type alpha motoneurons. In particular, the decreased succinate dehydrogenase activity of gamma motoneurons was observed only after long-term exposure to microgravity.     

Enzymatic coordination indices of leukocytes in the prognosis of mouse resistance to staphylococcal toxin

The activity of succinate, lactate and alpha-glycerophosphate dehydrogenases, as well as acid phosphatase, in the lymphocytes and neutrophils of the blood was studied in noninbred white mice prior to the intraperitoneal injection of 1 LD50 of staphylococcal toxin. As the result of intoxication, a half of the animals died and the other half survived. The two groups of the animals differed in the levels of the activity of succinate dehydrogenase and hyaloplasmatic alpha-glycerophosphate dehydrogenase in the neutrophils and lymphocytes, and also differed in the stability of correlations between the activity of succinate and lactate dehydrogenases in the lymphocytes and neutrophils and, besides, between the activity of lactate dehydrogenase in these types of cells.