scholarly journals Synthesis of adenosine triphosphate in isolated nuclei and intact cells

1967 ◽  
Vol 102 (3) ◽  
pp. 878-884 ◽  
Author(s):  
H. M. Klouwen ◽  
A. W. M. Appelman
Keyword(s):  
Oxidative Phosphorylation ◽  
Intact Cell ◽  
Atp Synthesis ◽  
Lymphoid Cells ◽  
Cell Nuclei ◽  
Isolated Cells ◽  
Intact Cells ◽  
Isolated Nuclei ◽  
Janus Green ◽  
Stimulation Of

1. It has previously been demonstrated that nuclei isolated from normal and neoplastic lymphoid cells are capable of oxygen-dependent ATP synthesis. In this paper it is shown that also the corresponding intact cells can synthesize ATP under those conditions in which nuclei can synthesize ATP. 2. In nuclei isolated from liver, kidney, rhabdomyosarcoma and osteosarcoma, oxygen-dependent ATP synthesis could not be demonstrated. The cells isolated from these tissues or tumours could not synthesize ATP either. The alternatives that such nuclei lost their ability for oxidative phosphorylation during the isolation procedure or that the process does not occur in these nuclei were explored. 3. Janus Green B, a vital stain for mitochondria, was used as a differential inhibitor of mitochondrial and nuclear ATP synthesis in intact cells. 4. Oxidative phosphorylation in mitochondria isolated from cells that had been incubated with various concentrations of Janus Green B (1-10mum) was seriously uncoupled, whereas at these concentrations oxygen-dependent ATP synthesis in isolated nuclei and in isolated cells were only inhibited to a small extent. 5. The results suggest that oxygen-dependent ATP synthesis in isolated cells measured under ;nuclear' conditions and in the presence of Janus Green B and Ca(2+) is mainly due to nuclear oxygen-dependent ATP synthesis. The stimulation of cellular ATP synthesis by glucose was completely inhibited by Janus Green B. 6. It is tentatively concluded that the stimulation of ATP synthesis in isolated cells by glucose, which is not found in isolated nuclei, represents mitochondrial ATP synthesis, and nuclear and mitochondrial ATP synthesis can then be studied differentially in the intact cell. The possibility is considered that oxygen-dependent nuclear ATP synthesis is not a general property of cell nuclei.

scholarly journals Role of nuclear glycogen synthase and cytoplasmic UDP glucose pyrophosphorylase in the biosynthesis of nuclear glycogen in HD33 Ehrlich-Lettré ascites tumor cells.

1981 ◽  
Vol 89 (3) ◽  
pp. 475-484 ◽  
Author(s):  
C Granzow ◽  
M Kopun ◽  
H P Zimmermann
Keyword(s):  
Tumor Cells ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Membrane System ◽  
Ascites Tumor ◽  
Cell Nuclei ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Isolated Nuclei ◽  
Glycogen Synthase Activity

Biochemical and autoradiographic evidence show both glycogen synthesis and the presence of glycogen synthase (UDP glucose [UDPG]: glycogen 4-alpha-D-glucosyltransferase; EC 2.4.1.11) in isolated nuclei of Ehrlich-Lettré mouse ascites tumor cells of the mutant subline HD33. 5 d after tumor transplantation, glycogen (average 5-7 pg/cell) is stored mainly in the cell nuclei. The activity of glycogen synthase in isolated nuclei is 14.5 mU/mg protein. At least half of the total cellular glycogen synthase activity is present in the nuclei. The nuclear glycogen synthase activity exists almost exclusively in its b form. The Km value for (a + b) glycogen synthase is 1 x 10(-3) M UDPG, the activation constant is 5 x 10(-3) M glucose-6-phosphate (Glc-6-P). Light and electron microscopic autoradiographs of isolated nuclei incubated with UDP-[1-3H]glucose show the highest activity of glycogen synthesis not only in the periphery of glycogen deposits but also in interchromatin regions unrelated to detectable glycogen particles. Together with earlier findings on nuclear glycogen synthesis in intact HD33 ascites tumor cells (Zimmermann, H.-P., V. Granzow, and C. Granzow. 1976. J. Ultrastruct. Res. 54:115-123), the results of tests on isolated nuclei suggest a predominantly appositional mode of nuclear glycogen deposition, without participation of the nuclear membrane system. In intact cells, synthesis of UDPG for nuclear glycogen synthesis depends on the activity of the exclusively cytoplasmic UDPG pyrophosphorylase (UTP: alpha-D-glucose-1-phosphate uridylyltransferase; EC 2.7.7.9). However, we conclude that glycogen synthesis is not exclusively a cytoplasmic function and that the mammalian cell nucleus is capable of synthesizing glycogen.

Regulation of oxidative phosphorylation in the mammalian cell

1990 ◽  
Vol 258 (3) ◽  
pp. C377-C389 ◽  
Author(s):  
R. S. Balaban
Keyword(s):  
Steady State ◽  
Energy Metabolism ◽  
Oxidative Phosphorylation ◽  
Atp Hydrolysis ◽  
Intact Cell ◽  
Classical Model ◽  
Atp Synthesis ◽  
Feedback Model ◽  
Feedback Network ◽  
The Individual

The cell is capable of maintaining a steady-state flux of energy from mitochondrial oxidative phosphorylation, producing ATP, to the cytosolic adenosinetriphosphatases (ATPases), performing work. Considerable effort has been devoted to investigating the individual mechanisms involved in these two processes. However, less effort has been directed toward learning how these reactions of energy metabolism interact through the cytosol to maintain the observed steady state in the intact cell. The "classical" model for the cytosolic interaction of these two processes involves the feedback of ATP hydrolysis products, ADP and Pi, from the ATPases to oxidative phosphorylation. This model is based on data from isolated mitochondria in which the rate of oxidative phosphorylation is controlled by the concentration of ADP and Pi. Yet, recent data from intact tissues with high oxidative phosphorylation capacities (i.e., heart, brain, and kidney) indicate that the cytosolic concentration of ADP and Pi do not change significantly with work. These data imply that this simple feedback model is not adequate to explain the regulation of energy metabolism in these tissues. Other sites within the oxidative phosphorylation process must be playing a regulatory role or the kinetics of ATP synthesis must be very different than currently believed to establish the steady state. This review covers the potential sites within oxidative phosphorylation which may be regulated through cytosolic transducers to result in the necessary feedback network regulating the steady-state flow of energy in the cell. These sites will include substrate delivery to the cytochrome chain, the processes involved in the phosphorylation of ADP to ATP, and the delivery of oxygen.

scholarly journals OXIDATIVE PHOSPHORYLATION AND ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA IN THE INTACT ASCITES TUMOR CELL

1971 ◽  
Vol 51 (1) ◽  
pp. 123-137 ◽  
Author(s):  
Charles R. Hackenbrock ◽  
Terry G. Rehn ◽  
Eugene C. Weinbach ◽  
John J. Lemasters
Keyword(s):  
Light Scattering ◽  
Oxidative Phosphorylation ◽  
Tumor Cell ◽  
Intact Cell ◽  
Adenine Nucleotides ◽  
Adenosine Diphosphate ◽  
Complete Inhibition ◽  
Ascites Tumor ◽  
Intact Cells ◽  
Ultrastructural Transformation

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90° light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox → condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90° light-scattering increase, which also occurs at this time, showed a t ½ of only 25 sec which agreed temporally with a slower orthodox → maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox → condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.

Oxidative stress: damage to intact cells and organs

1985 ◽  
Vol 311 (1152) ◽  
pp. 617-631 ◽  
Keyword(s):  
Cell Damage ◽  
Intact Cell ◽  
Feedback System ◽  
Transport Systems ◽  
Singlet Molecular Oxygen ◽  
Intact Cells ◽  
Extrahepatic Tissues ◽  
Stress Damage ◽  
Insight Into ◽  
Stimulation Of

Oxidative cell damage can be monitored by detection of ( a ) photoemission of singlet molecular oxygen formed from radical interactions (so-called low-chemical chemiluminescence), ( b ) end products of lipid peroxidation, such as ethane, and ( c ) glutathione disulphide release. These methods, preferably used in a complementary fashion, provide insight into the pro-oxidant-antioxidant balance in the intact cell or organ. Recent work from this laboratory on the metabolism of hydroperoxides and aldehydes as well as on redox cycling of the quinone menadione is presented. The comparison of GSSG transport systems in liver and heart reveals a limitation of capacity in the latter, thus making GSSG export potentially critical in the heart. As part of an inter-organ feedback system between extrahepatic tissues and liver, the newly described hormone stimulation of GSH release from liver is also presented.

The mitochondrial consequences of uncoupling intact cells depend on the nature of the exogenous substrate

2001 ◽  
Vol 355 (1) ◽  
pp. 231-235 ◽  
Author(s):  
Brigitte SIBILLE ◽  
Céline FILIPPI ◽  
Marie-Astrid PIQUET ◽  
Pascale LECLERCQ ◽  
Eric FONTAINE ◽  
...  
Keyword(s):  
Oxidation Rate ◽  
Marked Decrease ◽  
Electrical Potential ◽  
Atp Synthesis ◽  
Intact Cells ◽  
Isolated Mitochondria ◽  
Exogenous Substrate ◽  
High Oxidation ◽  
Transmembrane Electrical Potential ◽  
Sustained Increase

In isolated mitochondria the consequences of oxidative phosphorylation uncoupling are well defined, whereas in intact cells various effects have been described. Uncoupling liver cells with 2,4-dinitrophenol (DNP) in the presence of dihydroxyacetone (DHA) and ethanol results in a marked decrease in mitochondrial transmembrane electrical potential (∆ψ), ATP/ADP ratios and gluconeogenesis (as an ATP-utilizing process), whereas the increased oxidation rate is limited and transient. Conversely, when DHA is associated with octanoate or proline, DNP addition results in a very large and sustained increase in oxidation rate, whereas the decreases in ∆ψ, ATP/ADP ratios and gluconeogenesis are significantly less when compared with DHA and ethanol. Hence significant energy wastage (high oxidation rate) by uncoupling is achieved only with substrates that are directly oxidized in the mitochondrial matrix. Conversely in the presence of substrates that are first oxidized in the cytosol, uncoupling results in a profound decrease in mitochondrial ∆ψ and ATP synthesis, whereas energy wastage is very limited.

On the mechanism of ATP synthesis in oxidative phosphorylation: A review

1974 ◽  
Vol 3 (1) ◽  
pp. 1-15 ◽  
Author(s):  
John H. Young ◽  
Ephraim F. Korman ◽  
Jerome McLick
Keyword(s):  
Oxidative Phosphorylation ◽  
Atp Synthesis
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