RECOMBINANT HUMAN AND MOUSE PURPLE ACID-PHOSPHATASES - EXPRESSION ANDCHARACTERIZATION

The mammalian purple acid phosphatases (also called tartrate-resistant acid phosphatases) are expressed primarily in actively resorbing oste oclasts and activated macrophages, The enzymes are characterized by th e presence of a binuclear iron center at the active site. Recent studi es on transgenic mice lacking purple acid phosphatase implicate the os teoclast enzyme in both bone resorption and bone mineralization. To ch aracterize the mammalian enzymes in more detail, particularly with res pect to their substrate specificity at the low pH of the osteoclastic resorptive space (2.5-3), we have purified the recombinant human and m ouse enzymes from baculovirus-infected in. sect cells. The properties of the recombinant mouse enzyme are compared with those of the nonreco mbinant enzyme isolated from mouse spleen. The kinetics of hydrolysis of the substrates p-nitrophenyl phosphate, phosphotyrosine, and pyroph osphate and a phosphotyrosyl peptide by the recombinant human and mous e enzymes and the nonrecombinant mouse and pig enzymes were analyzed. For all the enzymes the ratio k(cat)/K-m was typically similar to 10(6 ) M-1 s(-1) and was higher at pH 2.5 than at 4.9. The increase was att ributable to a large decrease in K-m at the lower pH value. The result s indicate that the enzyme exhibits high catalytic efficiency toward s ubstrates such as pyrophosphate and acidic phosphotyrosine-containing peptides, particularly at low pH values typical of the bone resorptive space. The implications of the results for the physiological function of the enzyme are discussed. (C) 1997 Academic Press.