title: Comparative studies of rat recombinant purple acid phosphatase and bone tartrate-resistant acid phosphatase.
abstract: The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94%) identity at the amino acid sequence level with the purple acid phosphatase (PAP) from bovine spleen and with pig uteroferrin. These iron-containing purple enzymes contain a binuclear iron centre, with a tyrosinate-to-Fe(III) charge-transfer transition responsible for the purple colour. In the present study, production of rat osteoclast TRAP could be achieved at a level of 4.3 mg/litre of medium using a baculovirus expression system. The enzyme was purified to apparent homogeneity using a combination of cation-exchange, hydrophobic-interaction, lectin-affinity and gel-permeation chromatography steps. The protein as isolated had a purple colour, a specific activity of 428 units/mg of protein and consisted of the single-chain form of molecular mass 34 kDa, with only trace amounts of proteolytically derived subunits. The recombinant enzyme had the ability to dephosphorylate bone matrix phosphoproteins, as previously shown for bone TRAP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda max at 544 nm, which upon reduction with ascorbic acid changed to 515 nm, concomitant with the transition to a pink colour. EPR spectroscopic analysis of the reduced enzyme at 3.6 K revealed a typical mu-hydr(oxo)-bridged mixed-valent Fe(II)Fe(III) signal with g-values at 1.96, 1.74 and 1.60, proving that recombinant rat TRAP belongs to the family of PAPs. To validate the use of recombinant PAP in substituting for the rat bone counterpart in functional studies, various comparative studies were carried out. The enzyme isolated from bone exhibited a lower K(m) for p-nitrophenyl phosphate and was slightly more sensitive to PAP inhibitors such as molybdate, tungstate, arsenate and phosphate. In contrast with the recombinant enzyme, TRAP from bone was isolated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substituted with N-linked oligosaccharides. A slightly higher apparent molecular mass of the monomeric form and N-terminal chain of bone TRAP compared with the recombinant enzyme could not be accounted for by differential N-glycosylation. Despite differences in specific post-translational modifications, the recombinant PAP should be useful in future studies on the properties and regulation of the mammalian PAP enzyme.
authors.affiliation: Department of Immunology, Karolinska Institutet, Huddinge University Hospital, Sweden.
authors.forename: B
authors.initials: B
authors.lastname: Ek-Rylander
authors.forename: T
authors.initials: T
authors.lastname: Barkhem
authors.forename: J
authors.initials: J
authors.lastname: Ljusberg
authors.forename: L
authors.initials: L
authors.lastname: Ohman
authors.forename: K K
authors.initials: KK
authors.lastname: Andersson
authors.forename: G
authors.initials: G
authors.lastname: Andersson
mesh_terms: D000135:Acid Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001842:Bone and Bones / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007501:Iron / Q000032:analysis; D007527:Isoenzymes / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D013053:Spectrophotometry; D018411:Spodoptera / Q000235:genetics; D013329:Structure-Activity Relationship; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: D006023:Glycoproteins; D007527:Isoenzymes; D009844:Oligosaccharides; D010940:Plant Proteins; D011994:Recombinant Proteins; D007501:Iron; C091137:purple acid phosphatase; D010749:Phosphoprotein Phosphatases; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase