FLUORESCENT NUCLEOTIDE DERIVATIVES AS SPECIFIC PROBES FOR THE UNCOUPLING PROTEIN - THERMODYNAMICS AND KINETICS OF BINDING AND THE CONTROL BY PH

Fluorescent 2'-O-dansylated (DANS) purine nucleotides were synthesized . The fluorescence of the nucleotide derivatives is quenched in aqueou s solutions but strongly enhanced on binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria. The fluorescence enhanc ement was 30-, 10-, and 10-fold for DANSGTP, DANSATP, and DANSADP. One mole of DANS nucleotide binds to 1 mol of dimeric UCP. The binding af finity ranges from 10(5) to 10(8) M-l, similar to that of the unsubsti tuted nucleotides, while dansylation of AMP increases the affinity 50- fold. The pH dependence in the pK(D)/pH plots for the DANS nucleotides is basically similar to that for the unsubstituted nucleotides, i.e., for nucleoside diphosphates the slope Delta pK(D)/Delta pH < -1 at pH 5-6.5, = -1 at pH > 6.8, and only for triphosphates = -2 at pH > 7.2. Two different protonation sites with a pK(H) approximate to 4 (Asp/Gl u) and pK(H) approximate to 7.2 (His), only for nucleoside triphosphat es, are suggested to be involved in binding. The higher affinity of DA NSGTP indicates additional participation in binding of the C-6 oxygen on the guanine. The binding as measured with the anion exchange method agrees with the fluorescence measurement for DANSGTP, whereas for the more loosely binding DANSATP it is 40% lower. This is interpreted in terms of tight/loose UCP-nucleotide complexes, 100% tight complex for DANSGTP (as well GTP or ATP) but 40% loose complex for DANSATP. By mea suring the rapid kinetics using the fluorescence signal, the binding r ate is found to be fast and fairly constant for the various nucleotide s, whereas the dissociation is slow and strongly nucleotide dependent. The rates are pH dependent with Delta pk(on)/Delta pH = 1 for all the nucleotides and Delta pk(off)/Delta pH = -1 for DANSNTP but more weak ly with Delta pk(off)/Delta pH < -0.5 for DANSADP and DAN-ATP. The pH dependence of the binding rate corresponds to a protonation at the car boxylate group (Glu/Asp). The high pH dependence of the dissociation r ate only for DANSNTP is explained by deprotonation at the HisH(+) whic h is involved only in nucleoside triphosphate binding. This is in line with the very strong pH dependence of nucleoside triphosphate affinit y above pH 7 with a Delta pK(D)/Delta pH -2 as an important regulatory mechanism for the Hf transport activity of UCP. The differences of th e DANS nucleotides versus the DAN and unsubstituted nucleotides as wel l as the nucleoside tri- versus diphosphate are rationalized in a spec ific H+ dependent regulatory mechanism at the binding site.