[48] Isolation and hydrodynamic characterization of the uncoupling protein from brown adipose tissue

Klingenberg, Martin; Lin, Chi-shui

doi:10.1016/s0076-6879(86)26050-4

Cited by 12 publications

(6 citation statements)

References 16 publications

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“…Therefore, the experiments were repeated with the co-expressed AAC2 and His 6 -AAC2 in Triton X-100 and digitonin. Triton X-100 has been used previously for the solubilisation and purification of mitochondrial carriers 1,[23][24][25] and has been reported to preserve dimers of mitochondrial carriers in size-exclusion chromatography and analytical ultracentrifugation, 1,24,26 although we could not confirm the presence of dimers in an earlier study. 7 Digitonin is an extremely mild detergent, that has been used to examine the oligomeric state of the mitochondrial carriers by native gel electrophoresis.…”

Section: Transport Activity Of Single and Co-expressed Tagged And Untcontrasting

confidence: 65%

Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

Bamber

Slotboom

Kunji

2007

Journal of Molecular Biology

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Most mitochondrial carriers carry out equimolar exchange of substrates and they are believed widely to exist as homo-dimers. Here we show by differential tagging that the yeast mitochondrial ADP/ATP carrier AAC2 is a monomer in mild detergents. Carriers with and without six-histidine or hemagglutinin tags were co-expressed in defined molar ratios in yeast mitochondrial membranes. Their specific transport activity was unaffected by tagging or by co-expression. The co-expressed carriers were extracted from the membranes with mild detergents and purified rapidly by affinity chromatography. All of the untagged carriers were in the flow-through of the affinity column, whereas all of the tagged carriers bound to the column and were eluted subsequently, showing that stable dimers, consisting of associated tagged and untagged carriers, were not present. The specific inhibitors carboxyatractyloside and bongkrekic acid and the substrates ADP, ATP and ADP plus ATP were added during the experiments to determine whether lack of association might have been caused by carriers being prevented from cycling through the various states in the transport cycle where dimers might form. All of the protein was accounted for, but stable dimers were not detected in any of these conditions, showing that yeast ADP/ATP carriers are monomeric in detergents in agreement with their hydrodynamic properties and with their structure. Since strong interactions between monomers were not observed in any part of the transport cycle, it is highly unlikely that the carriers function cooperatively. Therefore, transport mechanisms need to be considered in which the carrier is operational as a monomer.

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Section: Transport Activity Of Single and Co-expressed Tagged And Untcontrasting

confidence: 65%

Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

Bamber

Slotboom

Kunji

2007

Journal of Molecular Biology

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“…Using quantitative Western blotting, the abundance of DIC was calculated to be 2.2 ± 0.4 mg l −1 culture of DIC‐pYES2 yeast strain and 60 ± 12 pmol mg −1 mitochondrial protein in the wild‐type strain (means ± SD of four determinations). Virtually all mitochondrial carrier proteins have been purified using hydroxyapatite as the major chromatographic step (for references see Palmieri, 1994), because they are not bound to hydroxyapatite in their native conformation (Klingenberg and Lin, 1986) in contrast to most other mitochondrial proteins. To test whether the overexpressed DIC retains the property of the native molecule to pass‐through hydroxyapatite, which also reflects the correct assembly in the mitochondrial membrane (Schleyer and Neupert, 1984; Zara et al ., 1991), we subjected the Triton X‐114‐solubilized mitochondria derived from the DIC‐pYES2 strain to hydroxyapatite chromatography.…”

Section: Resultsmentioning

confidence: 99%

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source

Palmieri

Vozza

Hönlinger

et al. 1999

Molecular Microbiology

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SummaryThe dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate-phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild-type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The ⌬DIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.

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“…The drastic effect of the pH of the medium on purine nucleotide binding has been shown first in isolated brown fat mitochondria at a pH range of pH 6.7-7.9 (Nicholls, 1976) and has been studied in detail over a large pH range in the isolated UCP (Lin and Klingenberg, 1982;Klingenberg, 1984: Klingenberg et al, 1986Klingenberg, 1988). In view of the different binding specificity of nucleotides interacting with UCP in the membrane and with the isolated protein, we controlled the pH dependence of purine nucleotide binding in urea particles.…”

Section: Discussionmentioning

confidence: 99%

“…The binding properties of solubilized mitochondrial carrier proteins depend on the detergent used for the protein extraction and may differ from those of the protein in the mitochondria (Lin and Klingenberg, 1982;Klingenberg, 1984 ;Weidemann et al, 1970). Solubilized UCP may be contaminated with other mitochondrial proteins (Klingenberg and Lin, 1986;Feil and Rafael, 1994) when isolated by the usual procedure (Lin and Klingenberg, 1982). This study reports on the interaction of nucleotides with UCP in mitochondrial membrane particles with free access of the ligands to all binding sites and no interference by other mitochondrial factors.…”

mentioning

confidence: 93%

Effect of pH and MgCl₂ on the binding of purine nucleotides to the uncoupling protein in membrane particles from brown fat mitochondria

Rafael

Pampel

Wang

1994

European Journal of Biochemistry

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Binding of purine nucleotides to the uncoupling protein (UCP) was investigated in membrane particles prepared from brown fat mitochondria of cold-acclimated rats. Mitochondrial membranes were separated from soluble protein with Lubrol WX and treated with 3 M urea at basic pH. The resulting membrane vesicles were permeable to GDP and contained up to 3 nmol UCP/mg protein with unchanged nucleotide binding, as compared to the mitochondria (GDP/UCP ratio = 1.0; pKd GDP = 6.0 at pH 7.0). UCP bound nucleotides to one type of specific binding sites, located exclusively on the cytosolic side of the mitochondrial membrane. The binding affinity of guanine nucleotides was 3-18-times higher than that of the corresponding adenine nucleotides, when measured in membrane particles from cold-acclimated rats, hamsters, and guinea pigs. The pH-dependent binding affinities of GDP and ADP attained a maximum at pH 5.0-6.0 (pKd GDP = 6.8, pKd ADP = 5.8) and were decreased by a factor of 10(2) at pH 4.0 and pH 8.0, respectively, whereas the binding affinity of ATP was maximal at pH 4.0 (pKd = 7.0) and was decreased by a factor of 10(3) at pH 7.5. Participation of the protein binding center in nucleotide interaction with UCP in the membrane was highly pH-dependent. Mg2+ modified the number of binding sites engaged at a given nucleotide concentration by complex binding of nucleotides; the Kd for Mg.GTP2- and Mg.GDP- was 20-50-times lower than that of the free nucleotides.

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[48] Isolation and hydrodynamic characterization of the uncoupling protein from brown adipose tissue

Cited by 12 publications

References 16 publications

Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source

Effect of pH and MgCl₂ on the binding of purine nucleotides to the uncoupling protein in membrane particles from brown fat mitochondria

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[48] Isolation and hydrodynamic characterization of the uncoupling protein from brown adipose tissue

Cited by 12 publications

References 16 publications

Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source

Effect of pH and MgCl2 on the binding of purine nucleotides to the uncoupling protein in membrane particles from brown fat mitochondria

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Effect of pH and MgCl₂ on the binding of purine nucleotides to the uncoupling protein in membrane particles from brown fat mitochondria