Energy coupling to sodium transport in frog skeletal muscle

1978 ◽  
Vol 235 (1) ◽  
pp. C25-C34 ◽  
Author(s):  
R. J. Connett ◽  
E. T. Hays
Keyword(s):  
Skeletal Muscle ◽  
Energy Production ◽  
Atp Hydrolysis ◽  
Energy Coupling ◽  
Creatine Phosphate ◽  
Coupling Factor ◽  
Metabolic Energy ◽  
Frog Skeletal Muscle ◽  
Energy Turnover ◽  
Sodium Efflux

In addition to a strophanthidin-sensitive (SS) sodium efflux, a large component of the sodium efflux in freshly isolated frog skeletal muscle is sodium-activated and strophanthidin-insensitive (SASI). The amount of metabolic energy associated with sodium movement by each of these components was measured and the coupling between sodium movement and adenosine 5'-triphosphate (ATP) hydrolysis in muscle was calculated. Energy production was blocked by iodoacetate and cyanide. Energy turnover was estimated from the change in creatine phosphate (CrP) and ATP contents and expressed as potential energy (PE = CrP + 2ATP). After metabolic poisoning a linear fall of PE occurred (6.3 mumol/g.h). Metabolic poisoning had no effect on the magnitude of the SS or SASI components of sodium efflux. In 2 h the sodium moved, and PE change due to the SS component was 4.35 and 1.66 mumol/g.h, respectively, which gave a coupling factor of 2.6. The amount of sodium moved by the SASI component was similar to that moved by the SS component in 2 h whereas no energy change was observed. It was, therefore, concluded that sodium movement by the SASI component requires no energy input.

scholarly journals Metabolic tracing reveals novel adaptations to skeletal muscle cell energy production pathways in response to NAD+ depletion

2019 ◽  
Vol 3 ◽  
pp. 147 ◽  
Author(s):  
Lucy A. Oakey ◽  
Rachel S. Fletcher ◽  
Yasir S. Elhassan ◽  
David M. Cartwright ◽  
Craig L. Doig ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Carbon Metabolism ◽  
Energy Production ◽  
Quantum Coherence ◽  
Metabolic Energy ◽  
Whole Body ◽  
Muscle Physiology ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Central Carbon ◽  
Metabolic Tracing

Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD+ availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD+ as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD+ homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD+ depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD+ metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U-13C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD+ excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD+ depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD+, which was rapidly reversed with repletion of the NAD+ pool using NR. NAD+ depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD+ depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD+ precursor usage in models of skeletal muscle physiology.

scholarly journals Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system

1987 ◽  
Vol 243 (2) ◽  
pp. 335-343 ◽  
Author(s):  
J M Fagan ◽  
L Waxman ◽  
A L Goldberg
Keyword(s):  
Skeletal Muscle ◽  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Gel Filtration ◽  
Metabolic Energy ◽  
Proteolytic System ◽  
Protein Conjugates ◽  
Erythroleukemia Cells ◽  
Endogenous Proteins ◽  
Fold Stimulation

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors.

scholarly journals In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Inda Setyawati ◽  
Weronika K Stanek ◽  
Maria Majsnerowska ◽  
Lotteke J Y M Swier ◽  
Els Pardon ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Energy Coupling ◽  
Coupling Factor ◽  
In Vitro System ◽  
In Vitro Reconstitution ◽  
Reconstituted System ◽  
Kinetics Of

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.

scholarly journals Metabolic tracing reveals novel adaptations to skeletal muscle cell energy production pathways in response to NAD+ depletion

2018 ◽  
Vol 3 ◽  
pp. 147 ◽  
Author(s):  
Lucy A. Oakey ◽  
Rachel S. Fletcher ◽  
Yasir S. Elhassan ◽  
David M. Cartwright ◽  
Craig L. Doig ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Carbon Metabolism ◽  
Energy Production ◽  
Quantum Coherence ◽  
Metabolic Energy ◽  
Whole Body ◽  
Muscle Physiology ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Central Carbon ◽  
Metabolic Tracing

Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD+ availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD+ as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD+ homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD+ depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD+ metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U-13C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD+ excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells results in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD+ depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity is increased in response to low NAD+, which is rapidly reversed with repletion of the NAD+ pool using NR. NAD+ depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reverses these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD+ depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD+ precursor usage in models of skeletal muscle physiology.

Ultra-Rapid Freezing of Isolated Skeletal Muscle Fibers

1977 ◽  
Vol 35 ◽  
pp. 576-577
Author(s):  
Joachim R. Sommer ◽  
Nancy R. Wallace
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Granular Material ◽  
Nuclear Envelope ◽  
Intracellular Calcium ◽  
Ruthenium Red ◽  
Threshold Concentration ◽  
Rapid Freezing ◽  
Frog Skeletal Muscle ◽  
Electrical Isolation

After Howell (1) had shown that ruthenium red treatment of fixed frog skeletal muscle caused collapse of the intermediate cisternae of the sarcoplasmic reticulum (SR), forming a pentalaminate structure by obi iterating the SR lumen, we demonstrated that the phenomenon involves the entire SR including the nuclear envelope and that it also occurs after treatment with other cations, including calcium (2,3,4).From these observations we have formulated a hypothesis which states that intracellular calcium taken up by the SR at the end of contraction causes the M rete to collapse at a certain threshold concentration as the first step in a subsequent centrifugal zippering of the free SR toward the junctional SR (JSR). This would cause a) bulk transport of SR contents, such as calcium and granular material (4) into the JSR and, b) electrical isolation of the free SR from the JSR.

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