Purple acid phosphatase of the human macrophage and osteoclast. Characterization, molecular properties, and crystallization of the recombinant di-iron-oxo protein secreted by baculovirus-infected insect cells

• Published in: The Journal of biological chemistry 1994-01, Vol.269 (2), p.1294-1300
• Main Authors: HAYMAN, A. R, COX, T. M
• Format: Article
• Language: English
• Subjects: Acid Phosphatase - chemistry; Acid Phosphatase - genetics; Analytical, structural and metabolic biochemistry; Animals; baculovirus; Biological and medical sciences; Cells, Cultured; Cloning, Molecular; Crystallography; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Genes; Humans; Hydrolases; In Vitro Techniques; Insecta; Luminescent Measurements; Macrophages - enzymology; Metalloproteins - chemistry; Moths; Nucleopolyhedroviruses - genetics; Osteoclasts - enzymology; Recombinant Proteins
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)42257-5

The purple phosphatases catalyze hydrolysis of phosphate esters (optimum pH approximately 5) and are resistant to inhibition by dextro-rotatory tartrate; their distinctive color is due to Fe(III)-phenolate charge-transfer transitions at their active site. Expression of human purple phosphatase, designated type 5 acid phosphatase, is restricted to osteoclasts and other activated cells of monohistiocytic lineage, but its biological rôle in relation to bone resorption and phagocytosis is unknown. To characterize this enzyme further, we have engineered the human type 5 acid phosphatase into a baculovirus vector expression system that enabled milligram quantities of purple protein to be purified from medium containing Sf9 host cells. The phosphatase cDNA was transcribed as a single RNA species of 1.5 kilobases as in human tissues. Tartrate-resistant acid phosphatase activity reacting with uteroferrin antisera appeared in the culture medium, from which up to 8 mg/liter was purified by two-step cation-exchange chromatography at pH 8.0. Two isoforms of approximately 36 kDa were identified by SDS-polyacrylamide electrophoresis and were converted to a single species of apparent molecular size 34 kDa upon treatment with N-glycosidase F, indicating secreted glycoforms of a single polypeptide. Mass spectroscopy showed that the mean molecular mass of the active, secreted glycoprotein was 35849 Da. The recombinant enzyme (specific activity, 190 mumol p-nitrophenol/min/mg at 37 degrees C) contained 2 iron atoms/molecule and formed purple, monoclinic crystals. Exposure to the ferric chelator, 1,2-dimethyl-3-hydroxypyrid-4-one, rapidly inactivated the enzyme, which was not inhibited by alpha, alpha'-bipyridyl, a ferrous chelator. That ferric iron is essential for enzymatic catalysis, was further indicated by the synergistic effects of the reductant, dithiothreitol, and bipyridyl on phosphatase activity. The recombinant purple phosphatase catalyzed the peroxidation of 5-aminophthalhydrazide (luminol), as evidenced by the induction of chemiluminescence; this reaction was inhibited by alpha, alpha'-bipyridyl at concentrations that did not inhibit phosphatase activity. The divalent iron moiety of human type 5 phosphatase may therefore participate in the generation of free radical species by fluid-phase reactions involving Fenton chemistry that are dissociated from its phosphatase function.