COMPARATIVE-STUDIES OF RAT RECOMBINANT PURPLE ACID-PHOSPHATASE AND BONE TARTRATE-RESISTANT ACID-PHOSPHATASE

The tartrate-resistant acid phosphatase (TRAP) of rat osteoclasts has been shown to exhibit high (85-94 %) identity at the amino acid sequen ce 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 tr ansition responsible for the purple colour. In the present study, prod uction of rat osteoclast TRAP could be achieved at a level of 4.3 mg/l itre of medium using a baculovirus expression system. The enzyme was p urified to apparent homogeneity using a combination of cation-exchange , hydrophobic-interaction, lectin-affinity and gel-permeation chromato graphy 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 proteolytica lly derived subunits. The recombinant enzyme had the ability to dephos phorylate bone matrix phosphoproteins, as previously shown for bone TR AP. Light absorption spectroscopy of the isolated purple enzyme showed a lambda(max) at 544 nm, which upon reduction with ascorbic acid chan ged to 515 nm, concomitant with the transition to a pink colour. EPR s pectroscopic analysis of the reduced enzyme at 3.6 K revealed a typica l 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 substitu ting for the rat bone counterpart in functional studies, various compa rative studies were carried out. The enzyme isolated from bone exhibit ed a lower K-m for p-nitrophenyl phosphate and was slightly more sensi tive to PAP inhibitors such as molybdate, tungstate, arsenate and phos phate. In contrast with the recombinant enzyme, TRAP from bone was iso lated predominantly as the proteolytically cleaved, two-subunit, form. Both the recombinant enzyme and rat bone TRAP were shown to be substi tuted with N-linked oligosaccharides. A slightly higher apparent molec ular mass of the monomeric form and N-terminal chain of bone TRAP comp ared with the recombinant enzyme could not be accounted for by differe ntial N-glycosylation. Despite differences in specific post-translatio nal modifications, the recombinant PAP should be useful in future stud ies on the properties and regulation of the mammalian PAP enzyme.