scholarly journals Changes in enzyme activities and distributions during glucose de-repression and respiratory adaptation of anaerobically grown Saccharomyces carlsbergensis

1973 ◽  
Vol 132 (3) ◽  
pp. 609-621 ◽  
Author(s):  
T. G. Cartledge ◽  
D. Lloyd
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiration Rate ◽  
Enzyme Activities ◽  
Specific Activity ◽  
Antimycin A ◽  
Saccharomyces Carlsbergensis ◽  
Whole Cells ◽  
Nadh Cytochrome ◽  
A Minor

1. During anaerobic glucose de-repression the respiration rate of whole cells of Saccharomyces carlsbergensis remained constant and was insensitive to antimycin A but was inhibited by 30% by KCN. Aeration of cells for 1 h led to increased respiration rate which was inhibited by 80% by antimycin A or KCN. 2. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucose de-repressed cells and the distribution of marker enzymes was investigated after zonal centrifugation on sucrose gradients containing MgCl2. These homogenates contained no detectable cytochrome c oxidase or catalase activity. The complex density distributions of NADH– and NADPH–cytochrome c oxidoreductases and adenosine triphosphatase(s) [ATPase(s)] were very different from those of anaerobically grown, glucose-repressed cells. 3. The specific activity of total ATPase was lowered and sensitivity to oligomycin decreased from 58 to 7% during de-repression. 4. Cytochrome c oxidase and catalase activities were detectable in homogenates of cells after 10min aeration. Zonal centrifugation indicated complex, broad sedimentable distributions of all enzyme activities assayed; the peaks of activity were at 1.27g/ml. 5. Centrifugation of homogenates of cells adapted for 30min and 3 h indicated a shift of density of the major sedimentable peak from 1.25g/ml (30min) to 1.235g/ml (3 h). After 30min adaptation a minor zone of oligomycin-sensitive ATPase and 15% of the total cytochrome c oxidase activities were detected at ρ=1.12g/l; these particles together with those of higher density containing cytochrome c oxidase, ATPase and NADH–cytochrome c oxidoreductase activities were all sedimented at 105g-min. 6. Electron microscopy indicated that the mitochondria-like structures of anaerobically grown, glucose-de-repressed cells were similar to those of repressed cells. After 10min of respiratory adaptation highly organized mitochondria were evident which resembled the condensed forms of mitochondria of aerobically grown, glucose-de-repressed cells. High-density zonal fractions of homogenates of cells after adaptation also contained numerous electron-dense vesicles 0.05–0.2μm in diameter. 7. The possibility that the `promitochondria' of anaerobically grown cells may not be the direct structural precursors of fully functional mitochondria is discussed.

scholarly journals Oscillations of enzyme activities during the cell-cycle of a glucose-repressed fission-yeast Schizosaccharomyces pombe 972h

1973 ◽  
Vol 136 (1) ◽  
pp. 195-207 ◽  
Author(s):  
R. K. Poole ◽  
D. Lloyd
Keyword(s):  
Cell Cycle ◽  
Cytochrome C ◽  
Schizosaccharomyces Pombe ◽  
Succinate Dehydrogenase ◽  
Cytochrome C Oxidase ◽  
Malate Dehydrogenase ◽  
Enzyme Activities ◽  
Nadh Dehydrogenase ◽  
Nadph Cytochrome ◽  
Nadh Cytochrome

1. Increased specific activities of cytochrome c oxidase, catalase, succinate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and malate dehydrogenase were observed during glucose de-repression of Schizosaccharomyces pombe. 2. The cell-cycle of this organism was analysed by three different methods: (a) harvesting of cells at intervals from a synchronous culture, (b) separation of cells by rate-zonal centrifugation into different size classes and (c) separation of cells by isopycnic-zonal centrifugation into different density classes. 3. Measurement of enzyme activities during the cell-cycle showed that all the enzymes assayed [cytochrome c oxidase, catalase, acid p-nitrophenylphosphatase, NADH-dehydrogenase, NADH–cytochrome c oxidoreductase, NADPH–cytochrome c oxidoreductase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase (NADP) and fumarate hydratase] show periodic expression as ‘peaks’. 4. Cytochrome c oxidase shows a single maximum at 0.67 of a cycle, whereas succinate dehydrogenase exhibits two maxima separated by 0.5 of a cell-cycle. 5. All other enzymes assayed showed two distinct maxima per cell-cycle; for catalase, malate dehydrogenase and NADPH–cytochrome c oxidoreductase there is the possibility of multiple fluctuations. 6. The single maximum of cytochrome c oxidase appears at a similar time in the cycle to one maximum of each of the other enzymes studied, except for NADH dehydrogenase. 7. These results are discussed with reference to previous observations on the expression of enzyme activities during the cell-cycle of yeasts.

Kinetics of the cytochrome c oxidase and reductase reactions in energized and de-energized mitochondria

1977 ◽  
Vol 55 (7) ◽  
pp. 706-713 ◽  
Author(s):  
Lars Chr. Petersen ◽  
Hans Degn ◽  
Peter Nicholls
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Concentration ◽  
Respiration Rate ◽  
Redox State ◽  
Rat Liver Mitochondria ◽  
Excess Oxygen ◽  
Succinate Oxidation ◽  
State Reduction

1. Coupled, cytochrome-c-depleted ('stripped') rat liver mitochondria reducing oxygen in the presence of exogenous cytochrome c, with succinate or ascorbate as substrates, show marked declines in the steady-state reduction of cytochrome c in excess oxygen on addition of uncouplers. Calculated ratios of maximal turnover in the uncoupled state and in the energized state for the cytochrome c oxidase (EC 1.9.3.1) reaction lie between 3 and 6, as obtained with reconstituted oxidase-containing vesicles. The succinate-cytochrome c reductase activity in such mitochondria shows a smaller response to uncoupler than that of the oxidase.2. The respiration rates of uncoupled mitochondria oxidizing ascorbate in the presence of added cytochrome c follow a Michaelis–Menten relationship with respect to oxygen concentration, in accordance with the pattern found previously with the solubilized oxidase. But succinate oxidation tends to give nonlinear concave-upward double-reciprocal plots of respiration rate against oxygen concentration, in accordance with the pattern found previously with intact uncoupled mitochondria.3. From simultaneous measurements of cytochrome c steady-state reduction, respiration rate, and oxygen concentration during succinate oxidation under uncoupled conditions it is found that at full reduction of cytochrome c, apparent Km for oxygen is 0.9 μM and the maximal oxidase (aa3) turnover is 400 s−1 (pH 7.4, 30 °C).4. The redox state of cytochrome c in uncoupled systems reflects a simple steady state; the redox state of cytochrome c in energized systems tends towards an equilibrium condition with the terminal cytochrome a3, whose apparent potential under these conditions is more negative than that of cytochrome c.

Endoplasmic reticulum membrane isolated from small-intestinal epithelial cells: enzyme and protein components

1981 ◽  
Vol 52 (1) ◽  
pp. 215-222
Author(s):  
M. Fujita ◽  
H. Ohta ◽  
T. Uezato
Keyword(s):  
Endoplasmic Reticulum ◽  
Epithelial Cells ◽  
Cytochrome C ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Cytochrome C Reductase ◽  
Basolateral Membranes ◽  
Nadh Cytochrome ◽  
Protein Components ◽  
A Minor

Endoplasmic reticulum membrane-rich fraction was obtained by subfractionation of the light microsomes from mouse jejunal mucosal epithelial cells. It was marked by high glucose-6-phosphatase, NADPH-cytochrome c reductase, and NADH-cytochrome c reductase activities and low Na+,K+-ATPase activity. The enrichment of Na+,K+-ATPase was 180-fold higher in the basolateral membranes than in the endoplasmic reticulum membrane-rich fraction relative to glucose-6-phosphatase. The protein peak that was phosphorylated in a Na-dependent manner was prominent in the basolateral membranes while it was a minor peak in the endoplasmic reticulum membrane-rich fraction. Under the electron microscope the fraction was seen to be composed of homogeneous small vesicles with thin smooth membranes.

scholarly journals Effects of L-Malate on Mitochondrial Oxidoreductases in Liver of Aged Rats

2011 ◽  
pp. 329-336 ◽  
Author(s):  
J.-L. WU ◽  
Q.-P. WU ◽  
Y.-P. PENG ◽  
J.-M. ZHANG
Keyword(s):  
Electron Transport ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Electron Transport Chain ◽  
Nadh Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Radical Scavenger ◽  
Aged Rats ◽  
Transport Chain ◽  
Nadh Cytochrome

Accumulation of oxidative damage has been implicated to be a major causative factor in the decline in physiological functions that occur during the aging process. The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS), considered as the pathogenic agent of many diseases and aging. L-malate, a tricarboxylic acid cycle intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production. Previous studies in our laboratory reported L-malate as a free radical scavenger in aged rats. In the present study we focused on the effect of L-malate on the activities of electron transport chain in young and aged rats. We found that mitochondrial membrane potential (MMP) and the activities of succinate dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats were significantly decreased when compared to young control rats. Supplementation of L-malate to aged rats for 30 days slightly increased MMP and improved the activities of NADH-dehydrogenase, NADH-cytochrome c oxidoreductase and cytochrome c oxidase in liver of aged rats when compared with aged control rats. In young rats, L-malate administration increased only the activity of NADH-dehydrogenase. Our result suggested that L-malate could improve the activities of electron transport chain enzymes in aged rats

Characterization of partially purified microbodies from hydrocarbon-grown cells of Cladosporium resinae

1989 ◽  
Vol 35 (5) ◽  
pp. 565-572 ◽  
Author(s):  
David B. Carson ◽  
Joseph J. Cooney
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Gradient Centrifugation ◽  
Urate Oxidase ◽  
Marker Enzyme ◽  
Thin Sections ◽  
Cell Extracts ◽  
Mitochondrial Fractions ◽  
A Minor

Cells of the filamentous fungus Cladosporium resinae synthesize many more microbodies when they are grown on an n-alkane than when they are grown on glucose. Cladosporium resinae was grown on n-dodecane and spheroplasts were prepared, disrupted, and fractionated by differential and density gradient centrifugation. A fraction was isolated which was enriched in catalase, a marker enzyme for microbodies. Another fraction was isolated which was enriched in cytochrome c oxidase, a marker for mitochondria. Urate oxidase, a second marker for microbodies, was not detected in cell extracts. The microbody and mitochondrial fractions were relatively free of contamination from the endoplasmic reticulum and cytosol as indicated by the amounts of glucose-6-phosphatase and glucose-6-phosphate dehydrogenase present, respectively. Transmission electron microscopy revealed that the catalase-enriched fraction contained intact microbodies, with mitochondria as a minor contaminant. Catalase was localized in microbodies by staining with 3,3′-diaminobenzidine. Mitochrondria were present in the cytochrome c oxidase enriched fraction and took up the vital stain Janus green B. In similar preparations from cells grown on glucose, catalase was largely nonparticulate. Microbodies were not observed in thin sections prepared from density gradient fractions, but mitochondria were present in a cytochrome c oxidase enriched fraction.Key words: Cladosporium resinae, microbodies, mitochondria, catalase, cytochrome c oxidase.

scholarly journals Oxidative phosphorylation during glycollate metabolism in mitochondria from phototrophic Euglena gracilis

1975 ◽  
Vol 150 (3) ◽  
pp. 373-377 ◽  
Author(s):  
N Collins ◽  
R H Brown ◽  
M J Merrett
Keyword(s):  
Cytochrome C ◽  
Oxygen Uptake ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Electron Acceptor ◽  
Euglena Gracilis ◽  
Gradient Centrifugation ◽  
Antimycin A ◽  
Isolated Mitochondria ◽  
Glycollate Dehydrogenase

Mitochondria were isolated by gradient centrifugation on linear sucrose gradients from broken cell suspensions of phototrophically grown Euglena gracilis. An antimycin A-sensitive but rotenone-insensitive glycollate-dependent oxygen uptake was demonstrated in isolated mitochondria. The partial reactions of glycollate-cytochrome c oxidoreductase and cytochrome c oxidase were demonstrated by using Euglena cytochrome c as exogenous electron acceptor/donor. Isolated mitochondria contain glycollate dehydrogenase and glyoxylate-glutamate aminotransferase and oxidize exogenous glycine. A P:O ratio of 1.7 was obtained for glycollate oxidation, consistent with glycollate electrons entering the Euglena respiratory chain at the flavoprotein level. The significance of these results is discussed in relation to photorespiration in algae.

scholarly journals Altered glycolytic and oxidative capacities of skeletal muscle contribute to insulin resistance in NIDDM

1997 ◽  
Vol 83 (1) ◽  
pp. 166-171 ◽  
Author(s):  
Jean-Aimé Simoneau ◽  
David E. Kelley
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Enzyme Activities ◽  
Glycolytic Enzymes ◽  
Oxidative Enzyme ◽  
Oxidative Enzymes ◽  
Glucose Tolerant

Simoneau, Jean-Aimé, and David E. Kelley. Altered glycolytic and oxidative capacities of skeletal muscle contribute to insulin resistance in NIDDM. J. Appl. Physiol. 83(1): 166–171, 1997.—The insulin resistance of skeletal muscle in glucose-tolerant obese individuals is associated with reduced activity of oxidative enzymes and a disproportionate increase in activity of glycolytic enzymes. Because non-insulin-dependent diabetes mellitus (NIDDM) is a disorder characterized by even more severe insulin resistance of skeletal muscle and because many individuals with NIDDM are obese, the present study was undertaken to examine whether decreased oxidative and increased glycolytic enzyme activities are also present in NIDDM. Percutaneous biopsy of vatus lateralis muscle was obtained in eight lean (L) and eight obese (O) nondiabetic subjects and in eight obese NIDDM subjects and was assayed for marker enzymes of the glycolytic [phosphofructokinase, glyceraldehyde phosphate dehydrogenase, hexokinase (HK)] and oxidative pathways [citrate synthase (CS), cytochrome- c oxidase], as well as for a glycogenolytic enzyme (glycogen phosphorylase) and a marker of anaerobic ATP resynthesis (creatine kinase). Insulin sensitivity was measured by using the euglycemic clamp technique. Activity for glycolytic enzymes (phosphofructokinase, glyceraldehye phosphate dehydrogenase, HK) was highest in subjects with subjects with NIDDM, following the order of NIDDM > O > L, whereas maximum velocity for oxidative enzymes (CS, cytochrome- c oxidase) was lowest in subjects with NIDDM. The ratio between glycolytic and oxidative enzyme activities within skeletal muscle correlated negatively with insulin sensitivity. The HK/CS ratio had the strongest correlation ( r = −0.60, P < 0.01) with insulin sensitivity. In summary, an imbalance between glycolytic and oxidative enzyme capacities is present in NIDDM subjects and is more severe than in obese or lean glucose-tolerant subjects. The altered ratio between glycolytic and oxidative enzyme activities found in skeletal muscle of individuals with NIDDM suggests that a dysregulation between mitochondrial oxidative capacity and capacity for glycolysis is an important component of the expression of insulin resistance.

scholarly journals Canine submandibular-gland hyaluronidase. Identification and subcellular distribution

1968 ◽  
Vol 110 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Y. H. Tan ◽  
J. M. Bowness
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Submandibular Gland ◽  
High Speed ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Product Formation ◽  
Triton X ◽  
Submandibular Saliva ◽  
Testicular Hyaluronidase

1. Submandibular glands from four species of mammal have been shown to contain a hyaluronidase active at acid pH; glands from dog and cat had a much higher content of this enzyme than has been found in other sources. 2. Product formation from hyaluronate after 24hr. incubation was almost the same as with testicular hyaluronidase, indicating that the enzyme is an endo-poly-β-hexosaminidase. 3. When submandibular-gland homogenates were fractionated by the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed, except cytochrome c oxidase, were found to occur partly in the soluble fraction and partly in the particulate fractions. Among the particular fractions, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome c oxidase, in the microsomal fraction for alkaline phosphatase and in the light-mitochondrial fraction for acid phosphatase, β-N-acetylhexosaminidase and acid-active hyaluronidase. 4. Release of the enzyme activity from the sedimentable fractions occurred in 0·1% Triton X-100 or after high-speed homogenization. 5. Stimulation of dogs by pilocarpine was found to decrease the hyaluronidase content of the submandibular gland by 5% and to cause the occurrence of a corresponding amount of acid-active hyaluronidase in the submandibular saliva. 6. The results are discussed in relation to the subcellular localization of hyaluronidase.

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