Activation of the multicatalytic proteinase from rat skeletal muscle by fatty acids or sodium dodecyl sulphate

A multicatalytic proteinase from rat skeletal muscle contains active site(s) catalysing the degradation of benzoyl-Val-Gly-Arg 4-methyl-7-coumarylamide, succinyl-Ala-Ala-Phe 4-methylcoumarylamide and [14C]methylcasein as well as benzyloxy-carbonyl-Leu-Leu-Glu 2-naphthylamide. These activities are 7-14-fold activated by 1 mM-sodium dodecyl sulphate. The activation leads to a higher susceptibility to the proteinase inhibitor chymostatin and to a lower ability to be inhibited and precipitated by antibodies raised against the non-activated enzyme. Since no changes in Mr or subunit composition were observed in the SDS-activated form, some conformational changes seem to occur during the activation step. More pronounced activation was observed in the presence of physiological concentrations of fatty acids; oleic acid at 100 microM concentrations stimulated the proteinase about 50-fold. In contrast with the non-activated proteinase, the activated enzyme considerably degrades muscle cytoplasmic proteins in vitro. Thus it is not unlikely that, in vivo, potential activators such as fatty acids can induce the multicatalytic proteinase to participate in muscle protein breakdown.

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Characterization of proteins from the cytoskeleton of Giardia lamblia

Journal of Cell Science ◽

10.1242/jcs.59.1.81 ◽

1983 ◽

Vol 59 (1) ◽

pp. 81-103 ◽

Cited By ~ 1

Author(s):

R. Crossley ◽

D.V. Holberton

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulphate ◽

Giardia Lamblia ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Molecular Size ◽

Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

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N tau-methylhistidine release: contributions of rat skeletal muscle, GI tract, and skin

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1982.243.4.e293 ◽

1982 ◽

Vol 243 (4) ◽

pp. E293-E297 ◽

Cited By ~ 2

Author(s):

S. J. Wassner ◽

J. B. Li

Keyword(s):

Skeletal Muscle ◽

Gastrointestinal Tract ◽

Soft Tissue ◽

Fractional Catabolic Rate ◽

Gi Tract ◽

Rat Skeletal Muscle ◽

Catabolic Rate ◽

Total Body

The relative contributions of skeletal muscle, gastrointestinal tract, and skin to urinary N tau-methylhistidine (MH) excretion were estimated during in vitro studies using the rat hemicorpus preparation. After 0.5 h of perfusion, MH release into the perfusate was linear for 3 h and averaged 29.8 nmol . h-1 . 100 g hemicorpus-1. In vivo, 24-h urinary MH excretion averaged 37.3 nmol . h-1 . 100 g body wt-1. The ratio of soft tissue to skin weight is equal (3.2:1) in the whole rat and in the hemicorpus. The gastrointestinal tract released 16.0 nmol . h-1 . 100 g body wt-1 or approximately 41% of the total urinary MH excretion. Preparations perfused with or without skin showed modest differences in the rate of MH release that were not statistically significant. Skeletal muscle contains 89.8% of total body MH content, whereas gastrointestinal tract and skin contain 3.8 and 6.4%, respectively. Gastrointestinal tract actomyosin turns over rapidly with a fractional catabolic rate of 24%/day versus 1.4%/day for skeletal muscle actomyosin.

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Effects of potassium deficiency on growth and protein synthesis in skeletal muscle and the heart of rats

British Journal Of Nutrition ◽

10.1079/bjn19890029 ◽

1989 ◽

Vol 62 (2) ◽

pp. 269-284 ◽

Cited By ~ 23

Author(s):

Inge Dôrup ◽

Torben Clausen

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Muscle Protein ◽

Control Level ◽

Inhibitory Effect ◽

Potassium Deficiency ◽

Skeletal Muscle Protein ◽

K Deficiency

The effects of potassium deficiency on growth, K content and protein synthesis have been compared in 4–13-week-old rats. When maintained on K-deficient fodder (1 mmol/kg) rats ceased to grow within a few days, and the incorporation of [3H]leucine into skeletal muscle protein in vivo was reduced by 28–38%. Pair-feeding experiments showed that this inhibition was not due to reduced energy intake. Following 14 d on K-deficient fodder, there was a further reduction (39–56 %) in the incorporation of [3H]leucine into skeletal muscle protein, whereas the incorporation into plasma, heart and liver proteins was not affected. The accumulation of the non-metabolized amino acid α-aminoisobutyric acid in the heart and skeletal muscles was not reduced. The inhibitory effect of K deficiency on 3H-labelling of muscle protein was seen following intraperitoneal (10–240 min) as well as intravenous (10 min) injection of [3H]leucine. In addition, the incorporation of [3H]phenylalanine into skeletal muscle protein was reduced in K-depleted animals. Following acute K repletion in vivo leading to complete normalization of muscle K content, the incorporation of [3H]leucine into muscle protein showed no increase within 2 h, but reached 76 and 104% of the control level within 24 and 72 h respectively. This was associated with a rapid initial weight gain, but normal body-weight was not reached until after 7 weeks of K repletion. Following 7 d on K-deficient fodder the inhibition of growth and protein synthesis was closely correlated with the K content of the fodder (1–40 mmol/kg) and significant already at modest reductions in muscle K content. In vitro experiments with soleus muscle showed a linear relationship between the incorporation of [3H]leucine into muscle protein and K content, but the sensitivity to cellular K deficiency induced in vitro was much less pronounced than that induced in vivo. Thus, in soleus and extensor digitorum longus (EDL) muscles prepared from K-deficient rats, the incorporation of [3H]leucine was reduced by 30 and 47 % respectively. This defect was completely restored by 24 h K repletion in vivo. It is concluded that in the intact organism protein synthesis and growth are very sensitive to dietary K deficiency and that this can only partly be accounted for by the reduction in cellular K content per se. The observations emphasize the need for adequate K supplies to ensure optimum utilization of food elements for protein synthesis and growth.

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The effect of sepsis in rats on skeletal muscle protein synthesis in vivo and in periphery and central core of incubated muscle preparations in vitro

Metabolism ◽

10.1016/0026-0495(88)90187-4 ◽

1988 ◽

Vol 37 (12) ◽

pp. 1120-1127 ◽

Cited By ~ 19

Author(s):

Robert P. Hummel ◽

Per-Olof Hasselgren ◽

J.Howard James ◽

Brad W. Warner ◽

Josef E. Fischer

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Central Core ◽

Skeletal Muscle Protein ◽

Skeletal Muscle Protein Synthesis

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PO-252 Effect of acupuncture intervention on the changes of cytoplasmic and mitochondrial Ca2+ concentration following eccentric contractions in rat skeletal muscle

Exercise Biochemistry Review ◽

10.14428/ebr.v1i5.11003 ◽

2018 ◽

Vol 1 (5) ◽

Author(s):

Aishan Liu ◽

Fangming Liu ◽

Xuelin Zhang ◽

Yarong Wang ◽

Mei Kong ◽

...

Keyword(s):

Skeletal Muscle ◽

Electrical Stimulation ◽

Fluorescent Imaging ◽

Eccentric Contractions ◽

Rat Skeletal Muscle ◽

Tetanic Force ◽

Acupuncture Intervention ◽

Stimulation Group

Objective The purpose of this study was to evaluate the effect of acupuncture intervention on the changes of cytoplasmic and mitochondrial Ca2+ concentration following eccentric contractions (ECC) in rat skeletal muscle. Methods 24 healthy male Wistar rats were randomly divided into 4 groups: control group (C, n=6)、electrical stimulation group (E, n=6)、electrical stimulation group with acupuncture intervention (EA, n=6)、electrical stimulation group with acupuncture +TRP channel inhibitor Gd3+ (EAI, n=6). The animal model of eccentric induced skeletal muscle injury was established by electrical stimulation on spinotrapezius muscle of anaesthetised rats in vivo, that is to say, the intact spinotrapezius muscle of adult Wistar rats was exteriorized, and tetanic eccentric contractions (100 Hz, 10 sets of 50 contractions) were elicited by electrical stimulation during synchronized muscle stretch of 10% resting muscle length. Cytoplasmic Ca2+ accumulation were determined by loading the muscle with fura 4-AM using fluorescent imaging in vivo, and mitochondrial Ca2+ concentration were determined by loading the muscle with fura 2-AM using fluorescent imaging in vitro, and recorded changes of muscle maximum tetanic force. Results (1) In vivo, compared with the C , cytoplasmic Ca2+ accumulation increased more rapidly during ECC in the E (P < 0.001). Acupuncture intervention significantly reduced cytosolic Ca2+ accumulation in the EA compared with the E (P < 0.01), and we discovered that muscle deformation generated by acupuncture intervention induced a robust Ca2+ spark response confined in close spatial proximity to the sarcolemmal membrane in intact muscle fibers. Although no significant differences between the EA and EAI, Gd3+ abolished the majority of cytoplasmic Ca2+ accumulation decrease during ECC in the EAI and a robust Ca2+ spark response disappeared compared with the EA. (2) In vitro, compared with the C, mitochondrial Ca2+ concentration did not elevations in MCC in the E. EA cytoplasmic Ca2+ increased rapidly above the C and E (P < 0.01), respectively, but EAI significantly attenuated the increases in mitochondrial Ca2+ concentration compared with the EA (P < 0.01). (3). Compared with the C , maximum tetanic force was significantly lower in the E after ECC (P < 0.01). EA maximum tetanic force increased rapidly compared with the E after ECC (P < 0.05), but EAL abolished the majority maximum tetanic force increase after ECC (P < 0.05). Conclusions (1)Eccentric contraction caused cytoplasmic Ca2+ accumulation, but mitochondrial Ca2+ concentration decrease. (2)Acupuncture can effectively reduce cytosolic Ca2+ overload, following by mitochondrial Ca2+ concentration increase , which in turn abnormally high cytoplasmic Ca2+ levels are buffed by the mitochondria, and improved muscle function, and the effect was associated to the TRP channels.

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PL - 033 A translational model of muscle protein synthetic bioactivity in vitro, ex vivo and in vivo

Exercise Biochemistry Review ◽

10.14428/ebr.v1i1.8513 ◽

2018 ◽

Vol 1 (1) ◽

Cited By ~ 1

Author(s):

Brian Carson ◽

Robert Davies ◽

Joseph Bass ◽

Catherine Norton ◽

Bijal Patel ◽

...

Keyword(s):

Skeletal Muscle ◽

Stable Isotope ◽

Resistance Exercise ◽

Ex Vivo ◽

Muscle Protein ◽

Muscle Growth ◽

Translational Model ◽

Nutrient Feeding

Objective The aim of this research was the development and validation of a translational model for the evaluation of exercise and nutrient stimulated muscle protein synthesis (MPS). To achieve this overall aim, three primary objectives had to be realised: (i) Development of an in vitro skeletal muscle cell bioassay to measure muscle growth and MPS; (ii) Development of an ex vivo model to evaluate the humoral effect on MPS in response to nutrient feeding and exercise; (iii) Use of a stable isotope technique to evaluate MPS in response to nutrient feeding and exercise in vivo. Methods To develop a novel in vitro skeletal muscle cell bioassay to measure muscle growth and MPS, C2C12 myoblasts were proliferated and subsequently differentiated to myotubes over 8 days in DMEM (2% HS). Changes in cell behavior and adhesion properties were monitored by measuring impedance via interdigitated microelectrodes using the xCELLigence system. MPS was measured by puromycin incorporation using the SUnSET technique, intracellular signalling measured by western blot, and myotube thickness by microscopy. To demonstrate the capability to monitor nutrient regulation of muscle growth, media was conditioned with a known potent regulator of MPS (leucine) in a dose response experiment (0.20 - 2.0 mM). To establish the ability of the bioassay to measure the humoral effect of MPS in response to feeding and exercise, media was conditioned by ex vivo human serum from fasted, rested, fed (protein and isonitrogenous non-essential amino acid (NEAA) control) and post-exercise conditions. To evaluate MPS in response to nutrient feeding and exercise in vivo, acute MPS (5 h) was assessed by measuring stable isotope deuterium oxide (D2O) incorporation into m. vastus lateralis skeletal muscle following consumption of either a Whey Protein (WP) or an isonitrogenous NEAA control combined with resistance exercise in resistance trained males. Results In vitro experiments observed a dose-response effect with a 32 % increase in cell index and a 27 % increase in cell thickness after 2 h in the presence of 2.0 mM leucine when compared with control myotubes. Ex vivo serum following ingestion of NEAA had no effect on protein signalling or MPS whereas WP fed serum significantly increased mTOR, P70S6K and 4E-BP1 phosphorylation (p<0.01, p<0.05) compared to fasted serum. Furthermore, the effect of WP fed serum on protein signalling and MPS was significantly increased (p<0.01, p<0.05) compared to NEAA fed serum. Ex vivo human serum following resistance exercise was also increased MPS (29 %) and phosphorylation of mTOR (6 %), p70S6K (12 %) and 4EBP1 (7 %), compared with resting serum. These ex vivo/in vitro findings translated to the in vivo model as myofibrillar fractional synthetic rates (myoFSR) (Basal 0.068±0.002%h-1 vs. WP 0.084±0.006 %h-1, p=0.033) and absolute synthetic rates (ASR) (Basal 10.34±1.01 vs. WP 13.18±0.71 g.day-1, p=0.026) were increased from basal levels only when resistance exercise was combined with WP ingestion and not the NEAA control (NEAA MPS 0.072±0.004%h-1, NEAA ASR 10.23±0.80 g.day-1). Thus, ingestion of WP in combination with resistance training augments acute MPS responses in resistance trained young men. Conclusions We have developed a translational model of muscle protein synthetic bioactivity using in vitro, ex vivo and in vivo methodologies. We have shown that we can impact MPS in vitro using ex vivo human serum to condition media, that MPS in vitro is differentially regulated by ex vivo serum containing bioactive WP compared to a non-bioactive NEAA control, and that this tranlates for resistance exercise combined with WP in humans when MyoFSR is measured using stable isotope technology. These experiments demonstrate that ex vivo/in vitro experiments translate to the in vivo model and these methods can be used to inform both exercise and nutrient human interventions.

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Skeletal muscle, liver and wool protein synthesis by sheep infected by the nematode Trichostrongylus colubriformis

Australian Journal of Agricultural Research ◽

10.1071/ar9751063 ◽

1975 ◽

Vol 26 (6) ◽

pp. 1063

Author(s):

LEA Symons ◽

WO Jones

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Muscle Protein ◽

Muscle Protein Synthesis ◽

Liver Protein ◽

Trichostrongylus Colubriformis ◽

Intestinal Nematode ◽

Skeletal Muscle Proteins

Incorporation of radioisotopically labelled L-leucine into skeletal muscle proteins was measured in vivo and in vitro, and into liver proteins in vivo in three groups of sheep: (1) infected by Trichostrongylus colubriformis, (2) uninfected, pair-fed with the infected animals, (3) uninfected, fed ad lib. Incorporation of [14C]L-leucine by an homogenate of wool follicles from infected and uninfected sheep was also measured. Incorporation of leucine by muscle, and hence muscle protein synthesis, was equally depressed in the anorexic infected sheep losing weight, and in pair-fed animals, whether measured in vivo or in vitro, or expressed in terms of either RNA or DNA. Incorporation into protein was elevated equally in vivo in the livers of the infected and pair-fed sheep when expressed in terms of content of tissue nitrogen, but not in terms of cither nucleic acid. Incorporation by the wool follicular homogenate was appreciably depressed by the infection and is consistent with the poor wool growth in nematode infections. These results show that the same depression of skeletal muscle and, possibly, elevation of liver protein synthesis occur in a ruminant as were reported earlier for laboratory monogastric animals with intestinal nematode infections. Pair-feeding uninfected animals in both this and the earlier experiments emphasized the importance of anorexia as a major cause of these effects on protein synthesis. The importance of these effects upon production is discussed briefly.

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Collagen fibril formation in the presence of sodium dodecyl sulphate

Biochemical Journal ◽

10.1042/bj2280551 ◽

1985 ◽

Vol 228 (3) ◽

pp. 551-556 ◽

Cited By ~ 20

Author(s):

G W Dombi ◽

H B Halsall

Keyword(s):

Conformational Changes ◽

Sodium Dodecyl Sulphate ◽

Collagen Fibril ◽

Hydrophobic Interactions ◽

Sodium Dodecyl ◽

Distal Region ◽

Fibril Formation ◽

Collagen Fibrillogenesis ◽

Fibril Morphology

Sodium dodecyl sulphate (SDS) was used to weaken both the electrostatic and the hydrophobic interactions during collagen fibrillogenesis in vitro. The rate and extent of fibril formation as well as fibril morphology were affected by SDS concentration. Both the formation of large fibrils at 0.3 mM-SDS and the complete cessation of fibril formation at 0.5 mM-SDS were considered to be the result of SDS-induced conformational changes in the non-helical telopeptides. A possible mechanism of SDS interaction with the N-terminal and the distal region of the C-terminal telopeptides is offered.

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