Regulation and organisation of the mitochondrial uncoupling protein from brown adipose tissue
Regulation and organisation of the mitochondrial uncoupling protein from brown adipose tissue
A specific immunoassay for uncoupling protein (UCP) and [3H]-GDP binding were used to investigate the time-course of the responses of brown adipose tissue (BAT) mitochondria, isolated from lean Zucker rats, to changes in environmental temperature. The level of GDP binding was shown to correlate with the proton-translocating activity of UCP (assessed by mitochondrial swelling). The chronic response involved synchronous changes in mitochondrial GDP binding and UCP concentration. However, the increased GDP binding observed upon acute cold-exposure (initial 4hr at 4oC) was not associated with any change in UCP concentration. The 'unmasking' of purine nucleotide binding sites on UCP was mimicked by acute administration of noradrenaline to both lean and obese Zucker rats. The reverse response ('remasking') was observed over the first 2 days of warm re-acclimation (27oC).
In order to probe the nature of the purine nucleotide binding site and the mechanisms of unmasking, specific modification and proteolytic studies were performed on UCP. Pyridoxal 5'-phosphate reduced the affinity of both the native and isolated protein for GDP, implying that a lysine residue(s) is involved in GDP binding. Tryptic and chymotryptic digestion of UCP within BAT mitochondria produced two fragments of 20-22,000 and 10-11,000 Mr. In the isolated protein, additional fragments were produced with chymotrypsin and also, if the pH was raised from 7.4 to 8.0, with trypsin. GDP was shown to have a protective effect on the high Mr fragment produced upon proteolysis of BAT mitochondria: GDP prevented further cleavage of this fragment to a product approximately 500-1,000 lower in Mr. 8-azido adenosine [γ-32P]triphosphate was used to specifically photolabel the purine nucleotide binding site on UCP. Digestion of the photolabelled protein within mitochondria and in the purified state, resulted in radioactivity only being incorporated into the 10-11,000 Mr fragment and the 10-15,000 Mr region respectively.
University of Southampton
Peachey, Tamsin Jane
c55ad4ff-ee33-496c-b90c-3bfb5315c6c3
1989
Peachey, Tamsin Jane
c55ad4ff-ee33-496c-b90c-3bfb5315c6c3
York, David
e5ba1de7-50b5-44ab-ad1b-485706ec369a
Peachey, Tamsin Jane
(1989)
Regulation and organisation of the mitochondrial uncoupling protein from brown adipose tissue.
University of Southampton, Doctoral Thesis, 230pp.
Record type:
Thesis
(Doctoral)
Abstract
A specific immunoassay for uncoupling protein (UCP) and [3H]-GDP binding were used to investigate the time-course of the responses of brown adipose tissue (BAT) mitochondria, isolated from lean Zucker rats, to changes in environmental temperature. The level of GDP binding was shown to correlate with the proton-translocating activity of UCP (assessed by mitochondrial swelling). The chronic response involved synchronous changes in mitochondrial GDP binding and UCP concentration. However, the increased GDP binding observed upon acute cold-exposure (initial 4hr at 4oC) was not associated with any change in UCP concentration. The 'unmasking' of purine nucleotide binding sites on UCP was mimicked by acute administration of noradrenaline to both lean and obese Zucker rats. The reverse response ('remasking') was observed over the first 2 days of warm re-acclimation (27oC).
In order to probe the nature of the purine nucleotide binding site and the mechanisms of unmasking, specific modification and proteolytic studies were performed on UCP. Pyridoxal 5'-phosphate reduced the affinity of both the native and isolated protein for GDP, implying that a lysine residue(s) is involved in GDP binding. Tryptic and chymotryptic digestion of UCP within BAT mitochondria produced two fragments of 20-22,000 and 10-11,000 Mr. In the isolated protein, additional fragments were produced with chymotrypsin and also, if the pH was raised from 7.4 to 8.0, with trypsin. GDP was shown to have a protective effect on the high Mr fragment produced upon proteolysis of BAT mitochondria: GDP prevented further cleavage of this fragment to a product approximately 500-1,000 lower in Mr. 8-azido adenosine [γ-32P]triphosphate was used to specifically photolabel the purine nucleotide binding site on UCP. Digestion of the photolabelled protein within mitochondria and in the purified state, resulted in radioactivity only being incorporated into the 10-11,000 Mr fragment and the 10-15,000 Mr region respectively.
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Peachey 1989 Thesis
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Published date: 1989
Identifiers
Local EPrints ID: 462700
URI: http://eprints.soton.ac.uk/id/eprint/462700
PURE UUID: 4b364377-8c15-46e7-9886-b2d827a39d3b
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Date deposited: 04 Jul 2022 19:42
Last modified: 20 Jun 2024 17:09
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Contributors
Author:
Tamsin Jane Peachey
Thesis advisor:
David York
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