Isolation of skeletal muscle mitochondria from hamsters using an lonic medium containing ethylenediarninetetraacetic acid and nagarse

Bhattacharya, Syamal K.; Thakar, Jay H.; Johnson, Patti L.; Shanklin, Douglas R.

doi:10.1016/0003-2697(91)90546-6

Cited by 92 publications

(47 citation statements)

References 11 publications

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“…The skeletal muscle was immediately dissected from the hindlimbs, weighed, and placed in isolation medium containing 100 mM KCl, 50 mM Tris-HCl, 2 mM EGTA, pH 7.4, on ice. Mitochondria were isolated from 4 -8 mice/preparation as described previously (34,35), with all steps at 4°C. Tissue was shredded with a sharp blade, minced with sharp scissors, rinsed with isolation medium four or five times, stirred for 2 min in a medium containing 100 mM KCl, 50 mM Tris-HCl, 2 mM EGTA, 1 mM ATP, 5 mM MgCl 2 , 0.5% (w/v) bovine serum albumin (BSA), and 18.7 units of protease/g of tissue (nagarse or Sigma protease type VIII), pH 7.4, and gently homogenized using a Polytron tissue homogenizer.…”

Section: Methodsmentioning

confidence: 99%

The Basal Proton Conductance of Skeletal Muscle Mitochondria from Transgenic Mice Overexpressing or Lacking Uncoupling Protein-3

Cadenas¹,

Echtay²,

Harper³

et al. 2002

Journal of Biological Chemistry

189

167

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The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was ϳ3 g/mg protein, ϳ20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and ϳ4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.

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Section: Methodsmentioning

confidence: 99%

The Basal Proton Conductance of Skeletal Muscle Mitochondria from Transgenic Mice Overexpressing or Lacking Uncoupling Protein-3

Cadenas¹,

Echtay²,

Harper³

et al. 2002

Journal of Biological Chemistry

189

167

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“…16 Liver mitochondria were isolated as previously described. 17 Rat skeletal muscle mitochondria were isolated according to the procedure of Bhattacharya et al 18 Rat hepatocytes were isolated according to the procedure of Seglen. 19 Whole rat brain synaptosomes were isolated according to Gordon-Weeks.…”

Section: Methodsmentioning

confidence: 99%

Indirect measurement of mitochondrial proton leak and its application

et al. 1999

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In mitochondria, ATP synthesis is coupled to oxygen consumption by the proton electrochemical gradient established across the mitochondrial inner membrane in a process termed oxidative phosphorylation. It has long been known from stoichiometric studies that ATP synthesis is not perfectly coupled to oxygen consumption. The major inef®ciency in the system is leakage of protons across the mitochondrial inner membrane driven by the proton electrochemical gradient. The kinetics of the proton leak can be determined indirectly, by measuring the oxygen consumption of mitochondria under non-phosphorylating conditions (plus oligomycin) as a function of the proton electrochemical gradient. This experimental system provides a convenient means to investigate inner membrane permeability to protons and the effect of factors that may effect that permeability. In this paper we review some results from our laboratory of indirect measurement of mitochondrial proton leak and how it has been applied to investigate the effect of aging, obesity and thyroid status on proton leak. The results show that (i) proton leak in isolated liver mitochondria is not signi®cantly different in a comparison of young and old rats, in contrast (ii) there is an apparent increase in proton leak in in situ mitochondria in hepatocytes from old rats when compared to those from young rats, (iii) proton leak in neuronal mitochondria in situ in synaptosomes is not signi®cantly different in young and old rats, (iv) proton leak is greater in isolated liver mitochondria from obaob mice compared to lean controls, (v) acute leptin (OB protein) administration restores the increased leak rate in isolated liver mitochondria from obaob mice to that of lean controls, (vi) administration of thyroid hormone (T 3 ) increases proton leak in rat muscle mitochondria, and (vii) proton leak in muscle mitochondria is insensitive to the presence of GDP. It is proposed that the experimental system described here for measuring proton leak, is an ideal functional assay for determining whether the novel uncoupling proteins increase inner membrane permeability to protons.

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“…Preparation of Mitochondria-Muscle mitochondria were prepared by the method modified from Bhattacharya et al (24). Muscle tissue (100 mg) was minced and incubated with 10% nagarse in ionic medium (100 mM sucrose, 10 mM EDTA, 100 mM Tris-HCl, 46 mM KCl, pH 7.4) for 5 min, washed with ionic medium containing 0.5% BSA, homogenized, and centrifuged (500 ϫ g, 10 min, 4°C).…”

Section: Study Of Mitochondrial Membrane Potential (⌬M)-c2c12 Cells (mentioning

confidence: 99%

Mitochondrial Alterations and Oxidative Stress in an Acute Transient Mouse Model of Muscle Degeneration

Ramadasan-Nair¹,

Gayathri

Mishra³

et al. 2014

Journal of Biological Chemistry

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Background: Human muscular dystrophies and inflammatory myopathies share common pathological events. Results: The cardiotoxin (CTX) model displayed acute and transient muscle degeneration and all the cellular events usually implicated in human muscle pathology. Conclusion: Mitochondrial alterations and oxidative stress significantly contribute to muscle pathogenesis. Significance: The CTX model is valuable in understanding the mechanistic and therapeutic paradigms of muscle pathology.

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Isolation of skeletal muscle mitochondria from hamsters using an lonic medium containing ethylenediarninetetraacetic acid and nagarse

Cited by 92 publications

References 11 publications

The Basal Proton Conductance of Skeletal Muscle Mitochondria from Transgenic Mice Overexpressing or Lacking Uncoupling Protein-3

The Basal Proton Conductance of Skeletal Muscle Mitochondria from Transgenic Mice Overexpressing or Lacking Uncoupling Protein-3

Indirect measurement of mitochondrial proton leak and its application

Mitochondrial Alterations and Oxidative Stress in an Acute Transient Mouse Model of Muscle Degeneration

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