Removal from the Membrane Affects the Interaction of Rat Osseous Plate Ecto-Nucleosidetriphosphate Diphosphohydrolase-1 with Substrates and Ions Daniela P. Garc ¸on Æ Douglas C. Masui Æ Rosa P. M. Furriel Æ Francisco A. Leone Received: 4 July 2008 / Accepted: 6 September 2008 / Published online: 8 October 2008 Ó Springer Science+Business Media, LLC 2008 Abstract We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane- bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site– site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucle- oside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min -1 mg -1 . The solubilized enzyme has M r of 66–72 kDa, and its catalytic efficiency was sig- nificantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always \ 2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic sig- naling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases. Keywords Osseous plate Á Endochondral ossification Á Digitonin Á Lysolecithin Á Suramin Á Ectonucleoside triphosphate diphosphohydrolase 1 Introduction Bone tissue formation is a complex biological process mediated by the concerted action of physicochemical and biochemical activities leading to the deposition of a min- eral phase into a specific organic matrix (Anderson 1995). The pioneering work of Robison (1923), who first associ- ated alkaline phosphatase activity with calcification, led to intensive investigations of the multiple activities of this enzyme (Anderson et al. 2005; Leone et al. 1997; Milla ´n 2006). However, it is now well established that alkaline phosphatase is not the sole enzyme in matrix vesicle membranes that may be relevant for calcification (Ander- son et al. 2005; Leone et al. 1997; Milla ´n 2006). Matrix membrane-invested vesicles are structures where the first crystals of calcium hydroxyapatite mineral are generated during biomineralization of growth plate carti- lage (Anderson et al. 2005). Mineralization of growing bone resembles that of growth plate cartilage in its initia- tion associated to submicroscopic, extracellular matrix vesicles. Although bone matrix vesicles are smaller and more rapidly calcified, they do resemble those of cartilage by being extracellular and not connected to cells (Anderson et al. 2005). The deposition of calcium phosphate also involves ATP-dependent transport of the cation into matrix vesicles (Ali and Evans 1973), thus suggesting the involvement of an ATPase in the mineralization process (Hsu and Anderson 1996; Pizauro et al. 1998). In spite of some circumstantial evidence, it remains unknown whether it is alkaline phosphatase or a specific ATPase that triggers the calcification of cartilage and bone since both enzymes can hydrolyze ATP (Anderson et al. 2005; Demenis and Leone 2000). The lack of substrate specificity and the multifunctional properties attributed to alkaline phospha- tase added to the difficulty of obtaining alkaline D. P. Garc ¸on Á D. C. Masui Á R. P. M. Furriel Á F. A. Leone (&) Departamento de Quı ´mica, Faculdade de Filosofia Cie ˆncias e Letras de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Avenida Bandeirantes 3900, 14040-901 Ribeira ˜o Preto, SP, Brazil e-mail: fdaleone@ffclrp.usp.br 123 J Membrane Biol (2008) 224:33–44 DOI 10.1007/s00232-008-9128-2