Separation and characterization of HeLa alkaline pyrophosphatase isoenzymes

Abstract

Three electrophoretic forms (isoenzymes) of alkaline pyrophosphatase from a human cell line HeLa Ch have been separated by preparative starch-gel electrophoresis followed by electrodialysis. One isoenzyme has an inorganic pyrophosphatase pH optimum of 8.8, whereas the other two isoenzymes have pyrophosphatase pH optima between 9.5 and 10.0. Both of the latter isoenzymes have inorganic pyrophosphatase activities which are inhibited by magnesium, whereas the pyrophosphatase activity of the protein with a pH optimum of 8.8 is not affected by this cation. All three proteins also possess magnesium-stimulated phosphomonoesterase activity with pH optima near 10. A purified preparation of these enzymes was used to study the substrate specificity as a pyrophosphatase. ADP and ATP, CDP and CTP, GDP and GTP, UDP, UTP, and ITP also functioned as substrates, in addition to PP i, whereas the biterminally substituted pyrophosphate NAD + did not serve as a substrate. Although both d- and l- cysteine inhibited the inorganic pyrophosphatase and phosphomonoesterase activities of these isoenzymes to the same extent, l-phenylalanine and l-tryptophan inhibited the pyrophosphatase activity less effectively than the phosphatase activity, suggesting interaction with an allosteric site. The molecular weights of these proteins are 100,000 as estimated by sucrose-density centrifugation and 150,000 as estimated by Sephadex G-200 chromatography.