8-bromoadenosine-5--triphosphate and 8-azidoadenosine-5--triphosphate

Numerous analytical experiments have shown that, in solution, ATP analogues with bulky substitutions at the eighth position of the adenine ring predominantly assume the syn conformation with respect to the adenine-ribose bond. Two such analogues, 3'-O-(N-methylanthraniloyl)-8-azido-ATP (Mant-8-N3-ATP) and 8-Br-ATP, were synthesized and used to probe the conformation of the ATP-binding site of myosin. In the presence of these analogues, actomyosin was rapidly dissociated; Mg2+-dependent ATP hydrolysis was significantly activated by actin; and Pi bursting was observed. For skeletal myosin, however, these analogues failed to support actin translocation, and they did not significantly enhance the intrinsic tryptophan fluorescence of skeletal muscle myosin subfragment-1 (SKE S-1). These results suggest that although myosin**/ADP/Pi intermediates can be formed with these analogues, the crucial conformational changes required for cross-bridge cycling do not occur in skeletal muscle myosin. The conformations of the ATP-binding sites of skeletal and smooth-muscle myosin were compared using the ternary complexes, myosin-ADP-beryllium fluoride (BeFn) or myosin-ADP-aluminium fluoride (AIF4-). In AlF4- complexes, Mant-8-N3-ADP affinity labeled the N-terminal 29-kDa domain of smooth-muscle myosin subfragment-1 (SM S-1), as did ATP analogues having the anti conformation, whereas it labeled the C-terminal 20-kDa domain of skeletal S-1. In smooth muscle BeFn complexes, Mant-8-N3-ADP was equally likely to cross-link to the 29-kDa N-terminal and the 25-kDa C-terminal domains. These analogues induced smooth muscle actomyosin super-precipitation and increased intrinsic tryptophan fluorescence to the same degree as ATP itself. As was expected from above results, the analogues supported smooth-muscle-myosin-induced actin translocation. These results suggest that smooth-muscle myosin adopts the eight-substituted ATP analogue in the normal conformation, but skeletal muscle myosin does not. This reflects the likely differences in the structures of their respective ATPase sites.

Topics: Adenosine; Adenosine Triphosphate; Animals; Azides; Binding Sites; Chickens; Hydrolysis; Light; Molecular Conformation; Molecular Probes; Muscle, Skeletal; Muscle, Smooth; Myosins; ortho-Aminobenzoates; Photoaffinity Labels; Rabbits; Scattering, Radiation; Spectrometry, Fluorescence

This paper describes a rapid and an efficient procedure for the enzymatic synthesis of 3'-phosphoadenosine 5'-phospho[35S]sulfate ([35S]PAPS). [35S]PAPS was synthesized by incubating ATP and a carrier-free [35S]-Na2(35)SO4 with ATP sulfurylase, a recombinant APS kinase and inorganic pyrophosphatase. The transfer of 35SO4 group from [35S]Na2SO4 to [35S]PAPS proceeded more efficiently in the presence of an ATP-regenerating system composed of pyruvate kinase and phosphoenol pyruvate. About 90% of the radioactivity present in the starting material [35S]Na2SO4 was transferred to [35S]PAPS within a 2-h reaction incubation. The reaction products were applied to a Mono Q column, and [35S]PAPS was eluted by a step-wise gradient of triethylamine bicarbonate buffer (pH 7.5). Under these conditions, [35S]PAPS eluted as a sharp peak at 0.7 M triethylammonium bicarbonate and it was very well separated from other contaminants. The purified [35S]PAPS (yield 85%, purity > 95%) was functional in donating sulfate to an oligosaccharide acceptor in a standard sulfotransferase reaction. The enzymatic procedure described above was particularly useful for the synthesis of [35S]PAPS at a wide range of concentrations and specific activities (up to 1500 Ci/mmol). This generally useful approach was also found to be successful in the syntheses of 8-azido and 8-bromo derivatives of [35S]PAPS. Applications of these two derivatives of PAPS, for purification and identification of sulfotransferases, have also been discussed.

Topics: Adenosine Triphosphate; Azides; Chromatography, High Pressure Liquid; Cloning, Molecular; Kinetics; Phosphoadenosine Phosphosulfate; Phosphotransferases (Alcohol Group Acceptor); Sulfate Adenylyltransferase