No abstract

Two substances, P1 and P2, have been isolated from the ammonium bicarbonate solutions of adenylylsulfate and 3′- phosphoadenylylsulfate, respectively. Using radiochemical, spectroscopic, chromatographic and enzymatic methods it could be shown that P1 was identical to adenosine 5′- monophosphoramidate and that P2 was a closely related phosphorylated derivative of the same substance, namely 3′-phosphoadenosine 5′-monophosphoramidate.

An improved enzymatic method for the synthesis of adenylylsulfate (APS) from adenosine 5'-phosphate using APS-reductase from Thiobacillus denitrificans is described. Isolation of millimole quantitities of this sulfur nucleotide is achieved rapidly by means of ion exchange chromatography on a strongly basic ion exchange resin. A facile and reproducible desalting procedure is described. Numerous procedures have been described for both the chemical and enzymatic synthesis and the isolation of APS*. De Meio [1] offers a review of these methods. The chromatographic procedures described thus far have proved to be unsatisfactory for routine preparative work in our laboratory. Ion exchange chromatography on Dowex 1 resin, the method originally used by Baddiley et al. [2], in volved large quantities of salt for elution. Desalting using activated charcoal and silica gel has proved, however, to be a tedious and unreliable procedure. Ion exchange chromatography using DEAE-cellulose and volatile buffers is now widely practiced and has proved to be an excellent method for the isolation of micromole amounts of this nucleotide. Prepara tion of millimole amounts of the nucleotide was, however, time-consuming, unwieldly and expensive due to the large columns and volumes of buffer needed for elution [3].It was therefore our goal to develop a rapid and inexpensive method for the isolation of millimole quantities of APS for research work in our labora tory. We sought to improve an existing enzymatic synthesis procedure and investigated the possibility of retaining Dowex 1 resin for ion exchange chromatography. Materials and MethodsThiobacillus denitrificans strain RT was obtained from M. Schedel (this institute) and grown accord ing to Baldensperger and Garcia [4]. The concentra * Abbreviation: Adenylylsulfate, APS; adenosine 5' monophosphate, AM P; adenosine 5'-diphosphate, ADP adenosine S'-triphosphate, ATP; DEAE-cellulose, diethy] aminoethyl-cellulose and EDTA, ethylenediametetraacetate.Reprint requests to Dr. B. P. Cooper. 0341-0382 / 79 / 0500-0346 $ 01.00/0 tions of Na2S20 3 and KN03 were raised to 50 m M and 60 m M , respectively, for large scale cultures. Cells were harvested after having reached an optical density of 1.3 (450 nm) by centrifugation in a Cepa continous flow centrifuge at 40,000 rpm, wash ed twice with 50 m M Tris-HCl buffer, pH 7.5, and finally suspended in the same buffer (20% w/v). If not used immediately the cell suspension was stored at -18 °C. Cell-free extracts were obtained by passage of the cell suspension through an Aminco French pressure cell at 80 bars pressure and centrifu gation. The cell suspension could be stored frozen for several months with no loss of activity.APS was synthesized in preliminary work ac cording to Adams et al. [5]. The APS synthesis was finally modified as follows to obtained a higher yield. For each 100 ml final volume 0.5 mmol Na2AMP, 0.05 mmol EDTA and then 1 . 0 mmol Na2S03 were dissolved in 80 ml of 80 m M Tris-HCl, pH 7.5, and then tempered in a water bath at 30 °C. Ten...