Enzymatic sulfation of steroids: VI. A simple, rapid method for routine enzymatic preparation of 3′-phosphoadenosine-5′-phosphosulfate

✍ Scribed by Sanford S. Singer

• Publisher: Elsevier Science
• Year: 1979
• Tongue: English
• Weight: 406 KB
• Volume: 96
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(79)90550-5

✦ Synopsis

A rapid method for enzymatic preparation of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is described. The method uses rat liver as the source of the PAPS synthesizing enzymes, and requires 2 days for production of 98 to 151 pmol of purified coenzyme. Purification of crude PAPS begins with ion-exchange chromatography on Dowex 1 columns. Impure PAPS pools from Dowex columns contain 3.1-4.2 pmol of nucleotide/g liver originally used. The final purification step involves chromatography on Sephadex G-10 columns. The resultant purified PAPS (2.0-3.1 pmol/original g liver) is 99 2 2% pure. PAPS is stored at -20°C as 1.0-1.2 mM solutions in 1.0 mM tris buffer (pH 8.7). These solutions are stable for at least 4-5 weeks. The method could probably be scaled up 6 to 12-fold without increasing total preparation time by more than 24 h.