The synthesis of a protein-reactive ATP analog, S-dinitrophenyl-6-mercaptopurine riboside 5'-triphosphate, started from isopropylidated 6-mercaptopurine. After dinitrophenylation, the thioester riboside was phosphorylated with POCI, and the monophosphate was converted to the triphosphate by activation with the aid of phosphoric acid diphenylester chloride, and reaction with inorganic pyrophosphate. and y-positions, was used for covalent binding of the nucleotide to rabbit muscle actin. In preparation for this affinity labelling, the one superficial --SH group of the protein had been alkylated with N-ethylmaleimide. The nucleotide was able to depolymerize N-ethylmaleimide-F-actin, and became attached to the G-form at a ratio of 0.8 molecules per actin monomer. The product could be repolymerized to the F-form with liberation of inorganic phosphate. The product could not be depolymerized after that by ATP solutions at low ionic strength.The analog, labelled with 32P in the A single radioactively labelled peptide was discerned in fingerprints of a tryptic digest.Actin, one of the two major filamentous proteins of the contractile system of muscle, possesses an unusual polymerization mechanism. The monomer form (G-actin) is isolated from acetone dry powder by extraction with ATP solutions of very low ionic content, each protomer binding one molecule of the nucleotide. Upon addition of magnesium or other salts, the bound triphosphate is hydrolyzed to ADP, and the ADP-protein complex polymerizes in a typical cooperative reaction (Engel, J., private communication) to double-helical filaments (Factin), present in muscle cells.The protein, except for slow and probably unphysiological recycling between F and G-forms, does not function as an ATPase. The binding and cleavage of the triphosphate however strongly resembles an enzymatic mechanism, coming to a stop after the one bound nucleotide has been hydrolyzed, before or during polymerization. Eur. J. Biochem. 43 (1974)binding site by X-(carboxynitrophenyl)-6-mercaptopurine riboside phosphate [I]. A related compound, X-dinitrophenyl-6-mercaptopurine riboside triphosphate, has now been obtained as an ATP analog, its synthesis and reaction with rabbit muscle actin are described. MATERIALS AND METHODSThe 6-mercaptopurine ribonucleoside, thioinosine was obtained from Waldhof Chemie (Mannheim). The protecting isopropylidene group was attached to the ribose moiety by the procedure of Hampton and Magrath [2]. Triethyl phosphate was distilled under vacuum not more than three weeks prior to use and kept over molecular sieve pearls (Merck (30.).Tetrasodium [32P]pyrophosphate (99.9 o/o asp, carrierfree) was obtained from the Amersham Radiochemical Centre (England). The solution was passed over a column of polysterene-sulfonic acid cation exchanger (Lewatit, Bayer) in the pyridinium form. The eluate was brought to dryness in a rotatory evaporator and traces of water were removed by adding 5ml dry pyridine, finally dry benzene, several times and evaporation of the solvent under vacuum. ...
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