Chapter 26 Preparation and Characterization of Mitochondria and Submitochondrial Particles of Rat Liver and Liver-Derived Tissues

Pedersen, Peter L.; Greenawalt, John W.; Reynafarje, Baltazar; Hullihen, Joanne; Decker, Glenn L.; Soper, John W.; Bustamente, Ernesto

doi:10.1016/s0091-679x(08)62030-0

Cited by 433 publications

(194 citation statements)

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“…Mitochondria from rat pancreas were prepared as described in MacDonald et al (22). To prepare mitoplasts from liver mitochondria, the mitochondrial pellets were diluted to 100 mg/ml with H medium (75 mM sucrose, 225 mM D-mannitol, 50 mM HEPES, and 1 mM bovine serum albumin) (23). An equal volume of extraction mixture (12 mg/ml digitonin and 0.5 mg/ml bovine serum albumin) was added and diluted by a 3-fold volume of H medium and centrifuged at 12,500 rpm for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Branched-chain Amino Acid Metabolon

Islam

Nautiyal

Wynn

et al. 2010

Journal of Biological Chemistry

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The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain ␣-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain ␣-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain ␣-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5-phosphateBCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.It has been proposed that the enzymes that are responsible for catalyzing sequential reactions in several metabolic pathways are highly organized in supramolecular complexes termed metabolons (1). The concept of metabolic enzymes associating to form a supramolecular complex was hypothesized 50 years ago (2). Recently, Benkovic and co-workers (3) have shown the in vivo assembly of six proteins in the purine catabolic pathway to form a "purinosome." The formation of the purinosome appears to be dynamically regulated by stimulation of de novo purine biosynthesis in response to changes in purine levels. Another group (4) also has shown that glycolytic enzymes ("glycosome") are organized into complexes, and this assembly is regulated by the oxidation and phosphorylation states of the proteins. The advantages of such a supramolecular assembly include efficient channeling of substrates between enzymes in a pathway, regulating pathway flux by association and dissociation, and by targeting the assembly of the interacting proteins to the appropriate intracellular structures. Previously, using liver mitochondrial extracts and purified BCATm, 2 we showed that the first two enzymes in BCAA catabolism, mitochondrial branched-chain aminotransferase (BCATm) and branchedchain ␣-keto acid decarboxylase (E1) of the BCKDC, associate in vitro (5). It is not yet known if this BCAA metabolon forms in tissues expressing BCATm, and if other associated proteins are functionally involved in the metabolon.BCATs (mitochondrial and cytosolic isozymes) catalyze reversible transamination of BCAA...

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Section: Methodsmentioning

confidence: 99%

Branched-chain Amino Acid Metabolon

Islam

Nautiyal

Wynn

et al. 2010

Journal of Biological Chemistry

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“…14 Mitochondrial respiration was monitored polarographically by an oxygraph equipped with a Clark-type oxygen electrode (Gilson Medical Electronics, Middleton, WI, USA).…”

Section: Isolation Of Rat Liver Mitochondria and Mitochondrial Respirmentioning

confidence: 99%

(+)α-Tocopheryl succinate inhibits the mitochondrial respiratory chain complex I and is as effective as arsenic trioxide or ATRA against acute promyelocytic leukemia in vivo

et al. 2011

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The vitamin E derivative (þ)a-tocopheryl succinate (a-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RARa transgenic mice, we demonstrated that a-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that a-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, a-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for a-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy.

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“…Purified mitochondria were subfractionated into outer membrane, inner membrane and matrix using digitonin (Roche) and differential centrifugation. 41 DNA and RNA analysis. The Ant1 genotype of animals was determined by differential PCR.…”

Section: Aav-mediated Gene Transfer Of Ant In Mousementioning

confidence: 99%