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.

Cytoplasmic pool of histone H1 in mammalian cells

1990 ◽  
Vol 96 (3) ◽  
pp. 461-468
Author(s):  
J.S. Zlatanova ◽  
L.N. Srebreva ◽  
T.B. Banchev ◽  
B.T. Tasheva ◽  
R.G. Tsanev
Keyword(s):  
Mammalian Cells ◽  
Mouse Liver ◽  
Peptide Mapping ◽  
Gel Filtration ◽  
Histone H1 ◽  
Cell Populations ◽  
Protein Preparation ◽  
Whole Cells ◽  
Erythroleukemia Cells ◽  
Core Histones

Two types of cell populations, nondividing mouse liver cells and exponentially growing Friend erythroleukemia cells, were studied for the presence of a histone H1 pool in the cytoplasm. Purified cytoplasmic fractions were extracted with 5% perchloric acid and the resulting protein preparation was characterized by two types of electrophoresis, gel filtration, peptide mapping, ELISA and immunoblotting. The occurrence of significant quantities of H1 in isolated cytoplasmic fractions was confirmed by indirect immunofluorescence on whole cells. The existence of a cytoplasmic pool of H1 contrasts with the lack of detectable amounts of core histones in the cytoplasm. This indicates that the observed H1 pool is not just a reflection of its cytoplasmic synthesis but probably has some functional significance.

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 Specificity of a protein phosphatase inhibitor from rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 435-444 ◽  
Author(s):  
P Cohen ◽  
G A Nimmo ◽  
J F Antoniw
Keyword(s):  
Skeletal Muscle ◽  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Glycogen Synthase ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Specific Inhibitor ◽  
Phosphorylase Kinase ◽  
Phosphatase Inhibitor ◽  
Protein Phosphatase Inhibitor

A hear-stable protein, which is a specific inhibitor of protein phosphatase-III, was purified 700-fold from skeletal muscle by a procedure that involved heat-treatment at 95 degrees C, chromatography on DEAE-cellulose and gel filtration on Sephadex G-100. The final step completely resolved the protein phosphatase inhibitor from the protein inhibitor of cyclic AMP-dependent protein kinase. The phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities of protein phosphatase-III [Antoniw, J. F., Nimmo, H. G., Yeaman, S. J. & Cohen, P.(1977) Biochem.J. 162, 423-433] were inhibited in a very similar manner by the protein phosphatase inhibitor and at least 95% inhibition was observed at high concentrations of inhibitor. The two forms of protein phosphatase-III, termed IIIA and IIIB, were equally susceptible to the protein phosphatase inhibitor. The protein phosphatase inhibitor was at least 200 times less effective in inhibiting the activity of protein phosphatase-I and protein phosphatase-II. The high degree of specificity of the inhibitor for protein phosphatase-III was used to show that 90% of the phosphorylase phosphatase and glycogen synthase phosphatase activities measured in muscle extracts are catalysed by protein phosphatase-III. Protein phosphatase-III was tightly associated with the protein-glycogen complex that can be isolated from skeletal muscle, whereas the protein phosphatase inhibitor and protein phosphatase-II were not. The results provide further evidence that the enzyme that catalyses the dephosphorylation of the alpha-subunit of phosphorylase kinase (protein phosphatase-II) and the enzyme that catalyses the dephosphorylation of the beta-subunit of phosphorylase kinase (protein phosphatase-III) are distinct. The results suggest that the protein phosphatase inhibitor may be a useful probe for differentiating different classes of protein phosphatases in mammalian cells.

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

Contraction duration affects metabolic energy cost and fatigue in skeletal muscle

1998 ◽  
Vol 274 (3) ◽  
pp. E397-E402 ◽  
Author(s):  
Michael C. Hogan ◽  
Erica Ingham ◽  
S. Sadi Kurdak
Keyword(s):  
Skeletal Muscle ◽  
Short Duration ◽  
Energy Cost ◽  
Lactate Concentration ◽  
Metabolic Energy ◽  
Muscle Lactate ◽  
Contracting Muscle ◽  
Contraction Duration ◽  
Long Duration ◽  
The Difference

It has been suggested that during a skeletal muscle contraction the metabolic energy cost at the onset may be greater than the energy cost related to holding steady-state force. The purpose of the present study was to investigate the effect of contraction duration on the metabolic energy cost and fatigue process in fully perfused contracting muscle in situ. Canine gastrocnemius muscle ( n = 6) was isolated, and two contractile periods (3 min of isometric, tetanic contractions with 45-min rest between) were conducted by each muscle in a balanced order design. The two contractile periods had stimulation patterns that resulted in a 1:3 contraction-to-rest ratio, with the difference in the two contractile periods being in the duration of each contraction: short duration 0.25-s stimulation/0.75-s rest vs. long duration 1-s stimulation/3-s rest. These stimulation patterns resulted in the same total time of stimulation, number of stimulation pulses, and total time in contraction for each 3-min period. Muscle O2 uptake, the fall in developed force (fatigue), the O2 cost of developed force, and the estimated total energy cost (ATP utilization) of developed force were significantly greater ( P < 0.05) with contractions of short duration. Lactate efflux from the working muscle and muscle lactate concentration were significantly greater with contractions of short duration, such that the calculated energy derived from glycolysis was three times greater in this condition. These results demonstrate that contraction duration can significantly affect both the aerobic and anaerobic metabolic energy cost and fatigue in contracting muscle. In addition, it is likely that the greater rate of fatigue with more rapid contractions was a result of elevated glycolytic production of lactic acid.

scholarly journals Characterization and purification of human retinoic acid receptor-γ 1 overexpressed in the baculovirus-insect cell system

1992 ◽  
Vol 287 (3) ◽  
pp. 833-840 ◽  
Author(s):  
A P Reddy ◽  
J Y Chen ◽  
T Zacharewski ◽  
H Gronemeyer ◽  
J J Voorhees ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Binding ◽  
Mammalian Cells ◽  
Retinoic Acid Receptor ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Sf9 Cells ◽  
Acid Receptor ◽  
Gel Retardation

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

scholarly journals Conserved Motifs within Ebola and Marburg Virus VP40 Proteins Are Important for Stability, Localization, and Subsequent Budding of Virus-Like Particles

2009 ◽  
Vol 84 (5) ◽  
pp. 2294-2303 ◽  
Author(s):  
Yuliang Liu ◽  
Luis Cocka ◽  
Atsushi Okumura ◽  
Yong-An Zhang ◽  
J. Oriol Sunyer ◽  
...  
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Ebola Virus ◽  
Intracellular Localization ◽  
Gel Filtration ◽  
Point Mutations ◽  
Marburg Virus ◽  
Structure Stability ◽  
Wild Type ◽  
Virus Like Particles

ABSTRACT The filovirus VP40 protein is capable of budding from mammalian cells in the form of virus-like particles (VLPs) that are morphologically indistinguishable from infectious virions. Ebola virus VP40 (eVP40) contains well-characterized overlapping L domains, which play a key role in mediating efficient virus egress. L domains represent only one component required for efficient budding and, therefore, there is a need to identify and characterize additional domains important for VP40 function. We demonstrate here that the 96LPLGVA101 sequence of eVP40 and the corresponding 84LPLGIM89 sequence of Marburg virus VP40 (mVP40) are critical for efficient release of VP40 VLPs. Indeed, deletion of these motifs essentially abolished the ability of eVP40 and mVP40 to bud as VLPs. To address the mechanism by which the 96LPLGVA101 motif of eVP40 contributes to egress, a series of point mutations were introduced into this motif. These mutants were then compared to the eVP40 wild type in a VLP budding assay to assess budding competency. Confocal microscopy and gel filtration analyses were performed to assess their pattern of intracellular localization and ability to oligomerize, respectively. Our results show that mutations disrupting the 96LPLGVA101 motif resulted in both altered patterns of intracellular localization and self-assembly compared to wild-type controls. Interestingly, coexpression of either Ebola virus GP-WT or mVP40-WT with eVP40-ΔLPLGVA failed to rescue the budding defective eVP40-ΔLPLGVA mutant into VLPs; however, coexpression of eVP40-WT with mVP40-ΔLPLGIM successfully rescued budding of mVP40-ΔLPLGIM into VLPs at mVP40-WT levels. In sum, our findings implicate the LPLGVA and LPLGIM motifs of eVP40 and mVP40, respectively, as being important for VP40 structure/stability and budding.

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