Evidence for unmasking of rat brown-adipose-tissue mitochondrial GDP-binding sites in response to acute cold exposure. Effects of washing with albumin on GDP binding

Gribskov, Cynthia L.; Henningfield, Mary F.; Swick, Andrew G.; Swick, Robert W.

doi:10.1042/bj2330743

Cited by 41 publications

(19 citation statements)

References 25 publications

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“…Dissociation was then followed by addition of 10 pM and 1 mM GTP. [2,231) who interpreted this as high/low-affinity sites. 2B, the dissociation of the bound ATP was rather slow.…”

Section: Resultsmentioning

confidence: 99%

Nature of the Masking of Nucleotide-Binding Sites in Brown Adipose Tissue Mitochondria. Involvement of Endogenous Adenosine Triphosphate

Huang¹,

Klingenberg²

1995

Eur J Biochem

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Binding of the fluorescent nucleotide derivative 2'-O-dansyl GTP and purine nucleotides to brown adipose tissue mitochondria from hamster was studied. 2'-O-Dansyl GTP binds with enhanced fluorescence to the uncoupling protein (UCP) in the mitochondria, similar to the isolated protein. The fluorescence signal showed biphasic fast and slow increases. Treatment of the mitochondria with an anion exchanger (Dowex) increased the total fluorescence but decreased the slower phase. The biphasic fluorescence response was restored by incubation with only 1 microM ATP, indicating that residual bound ATP may be responsible for the observed slow phase. The binding of [14C]GTP and GDP also increased after Dowex treatment. The dissociation of bound [14C]ATP but not of bound [14C]ADP was slow and apparently limited the binding assays. Short incubation (5 min) resulted in a curvature of the Scatchard plot, where the 'high-affinity sites' correspond to the free UCP sites; GDP had apparently higher affinity than GTP. Dowex treatment and incubation for 60 min produced a more linear Scatchard plot. Under such conditions, one measures the maximal UCP-binding sites (1.2 mumol/g protein); GTP exhibited higher affinity (Kd = 0.64 microM) than GDP (Kd = 3.1 microM). Acute cold adaptation (40 min at 4 degrees C) of hamsters caused an increase by over 40% of [14C]GTP binding, as compared to the control warm-(28 degrees C)-adapted animals. Dowex treatment completely abolishes this unmasking/masking effect, where both mitochondria had identical binding capacity and affinity for GTP. The inhibition by purine nucleotides of H+ transport as measured by potassium-acetate-induced mitochondrial swelling was dependent on the incubation time. Diphosphates inhibited faster and triphosphates required longer incubation (10 min) but inhibited more strongly. A linear correlation between the mitochondrial swelling rate and GDP binding was observed for mitochondria with depleted endogenous ATP or with added ATP. These data indicate that residual bound ATP from the tissue is responsible for the masking phenomenon.

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“…Dissociation was then followed by addition of 10 pM and 1 mM GTP. [2,231) who interpreted this as high/low-affinity sites. 2B, the dissociation of the bound ATP was rather slow.…”

Section: Resultsmentioning

confidence: 99%

Nature of the Masking of Nucleotide-Binding Sites in Brown Adipose Tissue Mitochondria. Involvement of Endogenous Adenosine Triphosphate

Huang¹,

Klingenberg²

1995

Eur J Biochem

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“…There is evidence to suggest that the extent of GDP binding can be dissociated from the UCP content of BAT mitochondria (Desautels et al, 1978;Gribskov et al, 1986;Trayhurn et al, 1987;Milner et al, 1988;Swick & Henningfield, 1989), i.e. rapid changes in GDP binding have been measured without any apparent changes in the amount of UCP.…”

Section: Resultsmentioning

confidence: 99%

“…Maximal GDP binding was measured in each experimental group. At the beginning of the experiment for each group we determined that 10 ,M-GDP produced the maximum GDP binding assayed with 0.3 mg ofmitochondrial protein, in accordance with Gribskov et al (1986). Mitochondria (0.3 mg of protein) were incubated for 10 min at 30°C in 2 ml of buffer containing 100 mM-[U-14C]sucrose (6 kBq/ml), 10 1tM- [8-3H] GDP (ammonium salt) (10 kBq/ml), 20 mM-Tes, 1 mM-EDTA and 5 itM-rotenone.…”

Section: Dietsmentioning

confidence: 99%

Evidence for masking of brown adipose tissue mitochondrial GDP-binding sites in response to fasting in rats made obese by dietary manipulation. Effects of reversion to standard diet

Puigserver

Lladó

Palou

et al. 1991

Biochemical Journal

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A specific immunoassay of uncoupling protein (UCP) and measurement of GDP binding were used to study the chronic responses of brown adipose tissue (BAT) mitochondria from rats made obese by dietary means (cafeteria rats)-and from obese rats subsequently fed a standard diet (post-cafeteria rats). We studied the response to fasting in order to assess the masking/unmasking responses in these groups. These studies have shown the following. (1) In the obese rats (cafeteria and post-cafeteria) the chronic increase in mitochondrial-UCP concentration compared with controls parallels the increase in GDP binding. (2) In 24 h-fasted control rats the decrease in GDP binding is associated with a change in UCP concentration, but in fasting cafeteria and post-cafeteria obese rats the decrease in GDP binding is not associated with any change in UCP concentration. (3) Post-cafeteria obese rats showed increased GDP binding and higher UCP concentrations than the controls, but these values were less than in cafeteria obese rats. (4) Control rats at 8 months old showed greater GDP binding and had a higher UCP concentration than 11-month-old control rats. (5) The responses of GDP binding and UCP concentration to fasting in post-cafeteria obese rats were similar to those in cafeteria obese rats, suggesting that such abbreviations are related to the obese status itself rather than to the composition of the cafeteria diet. The evidence supports the hypothesis that the response of the cafeteria and post-cafeteria obese rats to fasting is associated with a masking of UCP, whereas with chronic manipulation of diet changes in UCP concentration predominate. INTRODUCTIONBrown adipose tissue (BAT) is now viewed as a major site of both non-shivering and diet-induced thermogenesis in small mammals (for reviews see Shrago & Strieleman, 1987; HimmsHagen, 1989). The capacity for heat production in BAT is attributed to the loosely coupled respiration that can occur via the proton conductance pathway in mitochondria. Protons (or OH-) can pass through the BAT mitochondrial inner membrane via uncoupling protein (UCP), thus bypassing ATP synthesis (for reviews see Nicholls & Locke, 1984;Nicholls et al. 1986). All BAT mitochondria contain UCP (also known as thermogenin), which is a tissue-specific protein (M, 32000) that acts as a 'proton short-circuit', controlling the permeability of the inner membrane to protons. UCP is thus the principal factor regulating the uncoupling of respiration within the brown adipocyte (Nicholls & Locke, 1984).UCP has a high affinity for the binding of purine nucleotides (Heaton et al., 1978), and the binding of [3H]GDP to BAT mitochondria is frequently used as an indicator of thermogenic activity. Whether GDP binding is in fact a direct measure of the UCP content of the mitochondria has been the subject of several conflicting reports. Some studies suggest that the ability of UCP to bind GDP can change rapidly in response to acute cold exposure, noradrenaline and pharmacological agents or dietary manipulations (Desautels et ...

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“…Animals should be sacrificed without altering the environmental temperature, since specific GDP-binding may change rapidly, when rats acclimated to 4 "C or 29 "C (thermoneutrality) are exposed to room temperature [43]. Scatchard plots have been suggested to indicate different types of nucleotidebinding sites in UCP [30]. According to our findings, evaluation of Scatchard plots by a ligand-binding analysis program best fitted a one-site model [22].…”

Section: Discussionmentioning

confidence: 99%

Effect of acclimation temperature on the concentration of uncoupling protein and GDP binding in rat brown fat mitochondria

Feil

Rafael

1994

European Journal of Biochemistry

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The effect of the acclimation temperature on the concentration of the uncoupling protein (UCP) and specific GDP binding in rat brown adipose tissue mitochondria was investigated. UCP was measured by competition ELISA using purified UCP as a standard and antiserum developed against the C-terminus of the protein. UCP was purified by means of specific polyclonal antibodies immobilized on protein-A-agarose. It represented 2.4% of the total protein in brown fat mitochondria from rats acclimated to 4°C and 1.1% in mitochondria from rats acclimated to 29°C. No UCP was found in liver mitochondria. The molar ratio of bound GDP and UCP was 0.5 in mitochondria from warmacclimated rats and 1 .O in cold-acclimated rats. The GDPKJCP ratio was increased from 0.5 to 1 . O after a 90-min exposure to 4°C of warm-acclimated rats; it was decreased from 1.0 to 0.5, when cold-acclimated rats were transferred to 29°C for 24 h. Treatment of mitochondrial membranes from warm-acclimated rats with 3 M urea at pH 10.0 increased the GDPAJCP ratio from 0.5 to 1.0. Specific GDP binding was a direct measure of the UCP concentration during maximally activated or inactivated thermogenesis in brown adipose tissue. We suggest that variable GDP binding reflects the functional activity of UCP based on different protein conformations.The uncoupling protein (UCP) [I] in brown adipose tissue mitochondria, also called thermogenin [2], allows for a transmembrane flow of protons and thus for a chemiosmotic short circuit that uncouples respiration from ATP synthesis and dissipates chemical energy into metabolic heat. Nucleotides like ATP, ADP, GTP and GDP, which are specifically bound by UCP, inhibit the proton flow and restore respiratory control in the isolated mitochondria (reviewed in [3, 41). Nucleotide interaction with UCP is also likely to regulate the protein function in vivo. UCP, isolated with Triton X-100, has been shown to bind one nucleotide molecule/protein dimer [5]. It is, however, unclear, if this relationship applies also to UCP in the mitochondrial membrane. The molar ratio of bound GDP and UCP in rat brown adipose tissue mitochondria reported in the literature varies extensively [6-111. The relationship is made even more unclear by the altered nucleotide binding observed in mitochondria with unchanged UCP concentrations in response to acute cold or other forms of stimulation of the sympathetic nervous system (reviewed in [12]). According to the latter finding, the stoichiometry of nucleotide binding by UCP corresponds to the thermogenic activity of brown adipose tissue and thus to the activity of UCP. From this point of view, knowledge of the exact relationship between UCP and specifically bound nucleotide seemed important with respect to the molecular mechanism of UCP function. It was the purpose of this study to clarify the stoichiometry of GDP binding by UCP in mitochondria from brown adipose tissue of rats exposed to varying environmental temperatures. MATERIALS AND METHODS AnimalsTen-week-old male Sprague Dawley rats were maintain...

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Evidence for unmasking of rat brown-adipose-tissue mitochondrial GDP-binding sites in response to acute cold exposure. Effects of washing with albumin on GDP binding

Cited by 41 publications

References 25 publications

Nature of the Masking of Nucleotide-Binding Sites in Brown Adipose Tissue Mitochondria. Involvement of Endogenous Adenosine Triphosphate

Nature of the Masking of Nucleotide-Binding Sites in Brown Adipose Tissue Mitochondria. Involvement of Endogenous Adenosine Triphosphate

Evidence for masking of brown adipose tissue mitochondrial GDP-binding sites in response to fasting in rats made obese by dietary manipulation. Effects of reversion to standard diet

Effect of acclimation temperature on the concentration of uncoupling protein and GDP binding in rat brown fat mitochondria

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