phosphorus-radioisotopes and adenylyl-(3--5-)-uridine-3--monophosphate

Purified diphtheria toxin from various sources contains tightly, but noncovalently, bound nucleotides, the major component of which is adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). We used ApUp radiolabeled with 32P to measure equilibrium dissociation constants (KD), and association and dissociation rate constants (k+1 and k-1, respectively) under various conditions. Diphtheria toxin bound 1 molar equivalent of ApUp, regardless of the temperature. Values of KD were 0.2 nM (25 degrees C) and 1.8 nM (37 degrees C) as determined by flow dialysis. No difference in KD was observed between the nicked and intact forms of toxin. The dissociation rate constant showed marked variation with temperature, ranging from 1.8 X 10(-4) s-1 at 5.5 degrees C (t 1/2 of the complex = 64 min) to 2.5 X 10(-2) s-1 at 25 degrees C (t 1/2 of the complex = 28 s). By contrast, k+1 varied only by a factor of 5 over the same temperature range (2.0 X 10(7) M-1 s-1 at 5.5 degrees C; 9.6 X 10(7) M-1 s-1 at 25 degrees C). The KD at 5.5 degrees C, calculated from the ratio of k-1/k+1, is 9 pM, which represents the strongest affinity of a dinucleotide for a protein ever reported. Affinity was maximal in the range of pH 6.5 to 7.1 and was sensitive to ionic strength. Thermodynamic parameters of the system were calculated.

Topics: Chemical Phenomena; Chemistry; Diphtheria Toxin; Hydrogen-Ion Concentration; Kinetics; Oligonucleotides; Oligoribonucleotides; Phosphorus Radioisotopes; Thermodynamics