Sg. Huang et M. Klingenberg, NATURE OF THE MASKING OF NUCLEOTIDE-BINDING SITES IN BROWN ADIPOSE-TISSUE MITOCHONDRIA - INVOLVEMENT OF ENDOGENOUS ADENOSINE-TRIPHOSPHATE, European journal of biochemistry, 229(3), 1995, pp. 718-725
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.