Recombinant human and mouse purple acid phosphatases: expression and characterization.

Abstract

The mammalian purple acid phosphatases (also called tartrate-resistant acid phosphatases) are expressed primarily in actively resorbing osteoclasts and activated macrophages. The enzymes are characterized by the presence of a binuclear iron center at the active site. Recent studies on transgenic mice lacking purple acid phosphatase implicate the osteoclast enzyme in both bone resorption and bone mineralization. To characterize the mammalian enzymes in more detail, particularly with respect to their substrate specificity at the low pH of the osteoclastic resorptive space (2.5-3), we have purified the recombinant human and mouse enzymes from baculovirus-infected insect cells. The properties of the recombinant mouse enzyme are compared with those of the nonrecombinant enzyme isolated from mouse spleen. The kinetics of hydrolysis of the substrates p-nitrophenyl phosphate, phosphotyrosine, and pyrophosphate and a phosphotyrosyl peptide by the recombinant human and mouse enzymes and the nonrecombinant mouse and pig enzymes were analyzed. For all the enzymes the ratio k(cat)/Km was typically approximately 10(6) M(-1) s(-1) and was higher at pH 2.5 than at 4.9. The increase was attributable to a large decrease in Km at the lower pH value. The results indicate that the enzyme exhibits high catalytic efficiency toward substrates 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.

• Authors: K Marshall, K Nash, G Haussman, I Cassady, D Hume, J de Jersey, S Hamilton
• Journal: Archives of biochemistry and biophysics (Arch Biochem Biophys) Vol. 345 Issue 2 Pg. 230-6 (Sep 15 1997) ISSN: 0003-9861 [Print] United States
• PMID: 9308894 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Isoenzymes
• Peptide Fragments
• Recombinant Proteins
• Acid Phosphatase
• Tartrate-Resistant Acid Phosphatase

Topics

• Acid Phosphatase (biosynthesis, genetics)
• Animals
• Humans
• Isoenzymes (biosynthesis, genetics)
• Kinetics
• Mice
• Nucleopolyhedroviruses (genetics)
• Oxidation-Reduction
• Peptide Fragments (chemistry)
• Recombinant Proteins (biosynthesis)
• Sequence Analysis
• Species Specificity
• Spodoptera (cytology, virology)
• Swine
• Tartrate-Resistant Acid Phosphatase