NATURE OF THE MASKING OF NUCLEOTIDE-BINDING SITES IN BROWN ADIPOSE-TISSUE MITOCHONDRIA - INVOLVEMENT OF ENDOGENOUS ADENOSINE-TRIPHOSPHATE

Binding of the fluorescent nucleotide derivative 2'-O-dansyl GTP and p urine nucleotides to brown adipose tissue mitochondria from hamster wa s studied. 2'-O-Dansyl GTP binds with enhanced fluorescence to the unc oupling protein (UCP) in the mitochondria, similar to the isolated pro tein. The fluorescence signal showed biphasic fast and slow increases. Treatment of the mitochondria with an anion exchanger (Dowex) increas ed the total fluorescence but decreased the slower phase. The biphasic fluorescence response was restored by incubation with only 1 mu M ATP , indicating that residual bound ATP may be responsible for the observ ed slow phase. The binding of [C-14]GTP and GDP also increased after D owex treatment. The dissociation of bound [C-14]ATP but not of bound [ C-14]ADP was slow and apparently Limited the binding assays. Short inc ubation (5 min) resulted in a curvature of the Scatchard plot, where t he 'high-affinity sites' correspond to the free UCP sites; GDP had app arently higher affinity than GTP. Dowex treatment and incubation for 6 0 min produced a more linear Scatchard plot. Under such conditions, on e measures the maximal UCP-binding sites (1.2 mu mol/g protein); GTP e xhibited higher affinity (K-d = 0.64 mu M) than GDP (K-d = 3.1. mu M). Acute cold adaptation (40 min at 4 degrees C) of hamsters caused an i ncrease by over 40% of [C-14]GTP binding, as compared to the control w arm-(28 degrees C)-adapted animals. Dowex treatment completely abolish es this unmasking/masking effect, where both mitochondria had identica l binding capacity and affinity for GTP. The inhibition by purine nucl eotides of H+ transport as measured by potassium-acetate-induced mitoc hondrial swelling was dependent on the incubation time. Diphosphates i nhibited faster and triphosphates required longer incubation (10 min) but inhibited more strongly. A Linear correlation between the mitochon drial swelling rate and GDP binding was observed for mitochondria with depleted endogenous ATP or with added ATP. These data indicate that r esidual bound ATP from the tissue is responsible for the masking pheno menon.