Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1646, Issue 1-2, Pages 216-225Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S1570-9639(03)00021-9
Keywords
phosphatidylinositol-specific phospholipase C; endochondral ossification; suramin; sodium azide; NTPDase1
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An ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1) activity present in alkaline phosphatase-depleted rat osseous plate membranes, obtained 14 days after implantation of demineralized bone particles in the subcutaneous tissue of Wistar rats, was characterized. At pH 7.5, NTPDase1 hydrolyzed nucleotide triphosphates at rates 2.4-fold higher than those of nucleotide diphosphates, while the hydrolysis of nucleotide monophosphates and non-nucleotide phosphates was negligible. NTPDase 1 hydrolyzed ATP and ADP following Michaelis-Menten kinetics with V = 1278.7 +/- 38.4 nmol Pi/min/mg and K-M = 83.3 +/- 2.5 muM and V = 473.9 +/- 18.9 nmol Pi/min/mg and K-M = 150.6 +/- 6.0 muM, respectively, but in the absence of magnesium and calcium ions, ATP or ADP hydrolysis was negligible. The stimulation of the NTPDasel by calcium (V = 1084.7 +/- 32.5 nmol Pi/min/mg; and K-M =377.8 +/- 11.3 muM) and magnesium (V = 1367.2 +/- 41.0 nmol Pi/min/mg and K-M = 595.3 +/- 17.8 muM) ions suggested that each ion could replace the other during the catalytic cycle of the enzyme. Oligomycin, ouabain, bafilomycin A(1), theophylline, thapsigargin, ethacrynic acid, P-1,P-5-(adenosine-5')-pentaphosphate and omeprazole had negligible effects on the hydrolysis of ATP and ADP by NTPDasel. However, suramin and sodium azide were effective inhibitors of ATP and ADP hydrolysis. To our knowledge this is the first report suggesting the presence of NTPDasel in rat osseous plate membranes. Considering that the ectonucleoside triphosphate diphosphohydrolase family of enzymes participates in many regulatory functions, such as response to hormones, growth control, and cell differentiation, the present observations raise interesting questions about the participation of this activity in the calcification process. (C) 2003 Elsevier Science B.V. All rights reserved.
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