Removal from the membrane affects the interaction of rat osseous plate ecto-nucleosidetriphosphate diphosphohydrolase-1 with substrates and ions.
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 ectonucleoside 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 significantly 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 signaling 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.
Garçon
Daniela P
DP
Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Ribeirão Preto, SP, Brazil.
Masui
Douglas C
DC
Furriel
Rosa P M
RP
Leone
Francisco A
FA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism*; D001081:Apyrase / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006132:Growth Plate / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry*; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000737:chemistry; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D013379:Substrate Specificity
D015703:Antigens, CD; D007477:Ions; D008244:Lysophosphatidylcholines; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001081:Apyrase; C067869:CD39 antigen; D008274:Magnesium; D002118:Calcium