High-throughput assay for inorganic pyrophosphatases using the cytosolic enzymes of Saccharomyces cerevisiae and human as an example

This paper describes the development of a new, malachite green based, enzym atic assay for the identification of specific inhibitors of inorganic pyrop hosphatase (iPPase) from Saccharomyces cerevisiae for antifungal drug disco very. The human iPPase was used as counterscreen. The coding regions of bot h enzymes were amplified, cloned into a vector providing a His-tag at the C -terminus, expressed in Escherichia coli, and purified by metal chelate aff inity chromatography. Since the complete human sequence had not been publis hed previously, the human iPPase was cloned on the basis of expressed seque nce tag data. The human sequence was confirmed and showed about 55% amino a cid identity with the yeast enzyme and 95% identity with an already publish ed bovine enzyme. Both recombinant iPPases were characterized with regard t o their biochemical properties, showing that the His-tag did not influence the specific activity, pH optimum, inhibitor profile, or dimerization. The enzyme activity was determined by quantifying released phosphate by complex formation with malachite green. The resulting complex was quantified spect rophotometrically. The assay was adapted to a microtiter plate format, Thus , it is possible to screen a large compound pool for iPPase inhibitors in a short period of time. (C) 2000 Academic Press.