Abstract
The membrane-bound enzyme responsible for the breakdown of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) has been purified 1, 900-fold from detergent-solubilized human placenta, using chromatographies on Con A-Sepharose, Blue Sepharose, AMP-Agarose, and Sepharose CL-6B, and sucrose density gradient centrifugation. The enzyme required Mg2+ and showed the optimum activity at pH 9.4. The preparation was free of alkaline phosphatase [EC 3. 1. 3. 1], phosphodiesterase [EC 3. 1. 4. 1], and 5'-nucleotidase [EC 3. 1. 3. 5] activities, which enabled investigation of the substrate specificity and kinetic properties of the enzyme without interference by secondary reactions due to the above activities.
The enzyme cleaved the pyrophosphate linkages of NAD and various sugar nucleotides and the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate (PNTP), as well as the phosphosulfate linkages of PAPS and its biosynthetic precursor, adenosine 5'-phosphosulfate (APS), with apparent Km values of 0.12-0.33mM. Relative activities towards PNTP and PAPS did not change during the purification procedures starting from the homogenate. This, together with the data of kinetic studies using two substrates simultaneously, led us to conclude that the activities towards all the substrates tested were due to one and the same nucleotide pyrophosphatase.
