Brown‐Adipose‐Tissue Mitochondria: Photoaffinity Labelling of the Regulatory Site of Energy Dissipation

Abstract

Brown-adipose-tissue mitochondria possess an energy-dissipating ion uniport which is inhibited by purine nucleotides. The regulatory nucleotides bind to a high-affinity site on the outer face of the inncr membrane which is independent of the adenine nucleotide translocator. A direct correlation between affinity for the regulatory site and ability to inhibit the ion uniport is demonstrated for a numbcr of nucleotidc analogues. 8-Azido-adenosine 5'-triphosphate, a photoaffinity label, also competes with GDP f o r thc binding site and induces respiratory control. 8-Azido-adenosine [~-"'P]tripliospl~ate was prepared and covalcntly bound to hamster brown-adipose-tissue niitochondria by neur-ultraviolet irradiation. Two major radioactive bands were identified of apparent molecular weight 30000 and 32000, representing 6'%, and 10~

Decreased guanine nuleotide binding and reduced equivalent production by brown adipose tissue in hypothalamic obesity

“…Mr known to be associated with thermogenesis [13,14]. When VMH-lesioned rats and control rats are cold-acclimated, the binding of GDP as well as the percentage of 32000 Mr polypeptide increase and the same values are obtained in both groups (table 1) indicating that for these parameters, VMHlesioned rats exhibit a normal response to cold acclimation.…”

Localization of the High‐Affinity ATP Site in Adenosine‐3′:5′‐monophosphate‐Dependent Protein Kinase Type I

“…ATP was synthesized as described previously [13]. [y-32P]n!ATP was then prepared by an enzymatic exchange between [32P]orthophosphate and the yphosphate of n:ATP by an adaptation of the method of Glynn and Chappell [14] as described by Heaton et al [15]. The reaction mixture contained 50 mM Tris-C1 (pH 8.0) 15 pM NAD, 5 mM MgCI2, 2 mM cysteine, 1 mM 3-phosphoglycerate, 0.5 mM potassium dihydrogenphosphate, 50 pCi [32P]~rthophosphate, 17 U/ml 3-phosphoglycerate kinase/glyceraldehyde-3-phosphate dehydrogenase (activity ratio 1 : 1) (Boehringer, Mannheim) with 2.5 mM n!ATP as the substrate, in a final volume of 0.1 ml.…”

Modulation of β‐oxidation and proton conductance pathway of brown adipose tissue in hypo‐ and hyperinsulinemic states

“…The GDP binding sites are increased in high thermogenic states like cold acclimation [ 181 or spontaneous overfeeding [ 191 and decreased in low thermogenic states like warmadaptation [20] or genetic or experimentally induced obesity [21,22]. The 32000 Mr protein is increased in cold acclimation [9,10,18] and decreased in warm adaptation [9,23] and in experimentally induced obesity associated with overfeeding [22]. In diabetes, a state where BAT thermogenesis is reduced [5], both GDP-binding and 32000 M, protein are significantly decreased as compared to the control which is another indication that these two parameters are indices of the thermogenic capacity of BAT.…”